*Article* **Lactoferrin Prevents Hepatic Injury and Fibrosis via the Inhibition of NF-**κ**B Signaling in a Rat Non-Alcoholic Steatohepatitis Model**

**Yoshinaga Aoyama 1,2, Aya Naiki-Ito 1,\*, Kuang Xiaochen 1, Masayuki Komura 1, Hiroyuki Kato 1, Yuko Nagayasu 1, Shingo Inaguma 1, Hiroyuki Tsuda 3, Mamoru Tomita 4, Yoichi Matsuo 2, Shuji Takiguchi <sup>2</sup> and Satoru Takahashi <sup>1</sup>**


**Abstract:** Non-alcoholic steatohepatitis (NASH) can cause liver cirrhosis and hepatocellular carcinoma (HCC), with cases increasing worldwide. To reduce the incidence of liver cirrhosis and HCC, NASH is targeted for the development of treatments, along with viral hepatitis and alcoholic hepatitis. Lactoferrin (LF) has antioxidant, anti-cancer, and anti-inflammatory activities. However, whether LF affects NASH and fibrosis remains unelucidated. We aimed to clarify the chemopreventive effect of LF on NASH progression. We used a NASH model with metabolic syndrome established using connexin 32 (Cx32) dominant negative transgenic (Cx32ΔTg) rats. Cx32ΔTg rats (7 weeks old) were fed a high-fat diet and intraperitoneally injected with dimethylnitrosamine (DMN). Rats were divided into three groups for LF treatment at 0, 100, or 500 mg/kg/day for 17 weeks. Lactoferrin significantly protected steatosis and lobular inflammation in Cx32ΔTg rat livers and attenuated bridging fibrosis or liver cirrhosis induced by DMN. By quantitative RT–PCR, LF significantly down-regulated inflammatory (*Tnf-α*, *Il-6*, *Il-18*, and *Il-1β*) and fibrosis-related (*Tgf-β1*, *Timp2*, and *Col1a1*) cytokine mRNAs. Phosphorylated nuclear factor (NF)-κB protein decreased in response to LF, while phosphorylated JNK protein was unaffected. These results indicate that LF might act as a chemopreventive agent to prevent hepatic injury, inflammation, and fibrosis in NASH via NF-κB inactivation.

**Keywords:** NASH; lactoferrin; fibrosis; hepatocarcinogenesis; connexin

#### **1. Introduction**

The development of non-alcoholic fatty liver disease (NAFLD) is associated with obesity and disorders of lipid metabolism in patients with metabolic syndrome. With a global increase in recent years of the obese population, the number of cases of NAFLD has also increased [1,2]. A global meta-analysis describes the prevalence of NAFLD worldwide, which is approximately 25%, with the highest rates of 31% and 32% occurring in South America and the Middle East, respectively [3]. The concept of NAFLD is a broad spectrum of disease, ranging from simple steatosis without inflammation to non-alcoholic steatohepatitis (NASH) with chronic progressive inflammation and fibrosis. Continuous inflammation produces abundant inflammatory cytokines and accumulates reactive oxygen species (ROS) in the liver, leading to fibrosis. Once fibrosis develops to bridge cirrhosis, they are irreversible and can develop into hepatocellular carcinoma (HCC), as occurs in various chronic liver diseases, such as alcohol-induced injury and viral hepatitis [1,4].

**Citation:** Aoyama, Y.; Naiki-Ito, A.; Xiaochen, K.; Komura, M.; Kato, H.; Nagayasu, Y.; Inaguma, S.; Tsuda, H.; Tomita, M.; Matsuo, Y.; et al. Lactoferrin Prevents Hepatic Injury and Fibrosis via the Inhibition of NF-κB Signaling in a Rat Non-Alcoholic Steatohepatitis Model. *Nutrients* **2022**, *14*, 42. https:// doi.org/10.3390/nu14010042

Academic Editor: Md Soriful Islam

Received: 18 November 2021 Accepted: 20 December 2021 Published: 23 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Connexin (Cx) is a component of gap junctions, which exist between cells and is responsible for the transfer of small molecules less than 1 kDa, such as second messengers, ions, and cell metabolites [5,6]. This cellular interaction is called gap junctional intercellular communication (GJIC) and contributes to maintain tissue homeostasis and control cell growth and differentiation [7,8]. Within the liver, Cx32 exists as a major gap junction protein of hepatocytes [9,10]. In particular, decreased expression of Cx32 is followed by the continued progression of chronic liver diseases, such as liver cirrhosis and HCC [11].

We previously assessed the function of Cx32 in liver diseases via the establishment of Cx32 dominant negative transgenic (Cx32ΔTg) rats with a dominant negative mutant of Cx32 controlled by an albumin (Alb) promoter [12]. Cx32ΔTg rats showed greatly decreased Cx32 expression localized at the membrane and depressed GJIC capacity in their hepatocytes, as well as a high susceptibility to chemical-induced hepatocarcinogenesis compared to wild-type (Wt) rats [13,14]. Cx32 is involved in not only carcinogenesis but also NASH. There was no difference in susceptibilities to hepatotoxicity and hepatocarcinogenesis in the Cx32ΔTg as compared to Wt rats in basal diet feeding without any chemical treatment [14]. However, dysfunction of Cx32 in Cx32ΔTg rats exacerbated hepatocyte injury, steatohepatitis, and fibrosis due to increased ROS levels in the NASH induced by the methionine-choline deficient diet (MCDD) [10]. The acceleration of NASH development by Cx32 inactivation was also observed in another model that was induced by a combination of a high-fat diet (HFD) plus dimethylnitrosamine (DMN) in Cx32ΔTg rats [15]. Comparing the two models, nuclear factor (NF)-κB was commonly activated with the up-regulation of inflammatory cytokines, such as tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β1 in NASH induced by Cx32 dysfunction. Therefore, NF-κB is one of the key contributors to the progression of NASH.

Lactoferrin (LF) is an 80-kDa iron-binding glycoprotein found in all exocrine fluids, including tears, sweat, and saliva, and is especially abundant in milk. It was first isolated and purified in 1960 and was involved in the promotion of iron absorption and lipid metabolism. LF has various physiological functions, including anti-bacterial, anti-fungal, anti-viral, anti-oxidant, anti-cancer, anti-inflammatory effects, which have been reported. We focused on LF as a suppressor of inflammation. A previous study indicated that bovine LF inhibited chronic inflammation in the lungs in a mouse cystic fibrosis model. Tanaka et al. reported that bovine LF improved colitis in a dextran sulfate sodium-induced colitis model in rats and mice due to a reduction in the inflammation level by LF correlated with a decrease in proinflammatory cytokines, such as TNF-α, IL-1β, and interleukin (IL)-6 [16,17]. With regard to the liver, decreased IL-1β by LF leads to the inhibition of carbon tetrachloride-induced hepatitis in a rat model [18]. Another report suggests that LF reduced the expression of TGF-β1, IL-1β, and TNF-α and suppressed liver fibrosis in a rat systemic lupus erythematosus model [19]. The anti-tumor abilities of LF have also been described in various cancer cell lines, such as those of the breast [20], stomach [21], head, and neck [22]. A randomized placebo-controlled clinical trial indicated that the growth of colorectal adenomatous polyp was significantly retarded by intake of 3 g LF without any adverse events related to the intervention [23]. However, the effects of LF on hepatotoxicity, as well as fibrosis and carcinogenesis on NASH, have not been clearly established as yet.

In this study, we aimed to determine the chemopreventive effect of dietary LF on NASH development and hepatocarcinogenesis using a Cx32ΔTg–HFD–DMN NASH model.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

A HFD (HFD-60) was bought from Oriental BioService, Inc. (Kyoto, Japan). DMN was supplied by Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan). Bovine LF was provided by the Dairy Techno Inc. (Tokyo, Japan).

#### *2.2. Development and Screening of Transgenic Rats*

Cx32ΔTg rats were bred and screened as previously described [12]. Rats were housed in cages containing hardwood chips under specific pathogen-free conditions at 22 ± 2 ◦C and 50% humidity using a 12 h light/12 h dark cycle. Rats ate food and tap water that were available ad libitum. Protocols for animal experiments were approved by the Institutional Animal Care and Use Committee of Nagoya City University School of Medical Sciences (no. 19-025, approved on 24 September 2019).

#### *2.3. Animal Treatments and Biochemical Analysis*

A total of 48 male Cx32ΔTg rats (7 weeks old) ate a HFD for 17 weeks. After 5 weeks, DMN was injected intraperitoneally six times once every 2 weeks. DMN was used at 15 mg/kg (injections 1 and 2), 10 mg/kg (injections 3 and 4), and 5 mg/kg (injections 5 and 6). Rats were randomly divided into three groups (*n* = 16 each). One group of rats received tap water (Control), and the other two groups of rats continuously received either 100 or 500 mg/kg/day LF (LF100 or LF500) in drinking water for 17 weeks. During animal experiments, one rat in the control group unexpectedly died at week 10. Therefore, we analyzed 47 rats in total (Control: 15 rats, LF100: 16 rats, and LF500: 16 rats) once the experiment was completed. All rats were sacrificed under deep anesthesia, and samples of blood were taken from the abdominal aorta. Total adipose tissues around spermatic ducts were weighed to assess visceral fat.

The serum levels of Alb, total protein, alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol (LDL-C), total cholesterol, and glucose were assessed and measured at the DIMS Institute of Medical Science, Inc. (Aichi, Japan).

#### *2.4. Histology of NASH*

Rat livers were surgically excised and sliced into 3–4 mm thick sections. After fixing with 10% buffered formalin, sections were embedded in paraffin for histological evaluation (thickness 2–3 μm). Histological sections were stained with Azan or hematoxylin and eosin (H&E), as well as immunohistochemically stained with antibody against α-smooth muscle actin (α-SMA; Dako, Tokyo, Japan). Steatohepatitis and fibrosis were evaluated using a non-alcoholic fatty liver disease activity score (NAS), as previously described in detail [10,24]. The NAS and fibrosis scores were evaluated by three very experienced pathologists (A.N.-I., M.K., and S.Takahashi).

#### *2.5. Evaluation of Preneoplastic Foci in the Liver*

The glutathione S-transferase placental form (GST-P) was immunohistochemically stained, as previously described [25]. Averages of GST-P–positive foci that were >80 μm in diameter in the entire liver section were evaluated using an image analyzer (Keyence, Osaka, Japan).

#### *2.6. Western Blotting*

Protein samples were extracted from frozen liver tissues using radioimmunoprecipitation buffer (Thermo Fisher Scientific, Rockford, IL, USA) with added protease and phosphatase inhibitors (Thermo Fisher Scientific). The protein concentration of samples was quantified by a Bradford assay. Protein samples (30 μg per lane) were separated in 12% polyacrylamide gels and transferred onto nitrocellulose membranes (Hybond-ECL; GE Healthcare UK Ltd., Buckinghamshire, UK). Membranes were probed with primary antibodies against: Cdc42, IκB-α, NF-κB, phosphorylated (p) NF-κB (Ser536), Mkk4, pMkk4 (Thr261), Jnk, pJnk (Thr183/Tyr185) (Cell Signaling Technology, Danvers, MA, USA), and β-actin (Sigma-Aldrich, St. Louis, MI, USA). Anti–β-actin was used at a 1:5000 dilution, and all other antibodies were used at 1:1000. ImageJ software, ver.1.52 (National Cancer Institute, Bethesda, MD, USA), was used to quantify bands from blots.

#### *2.7. Quantitative Reverse Transcription PCR*

RNA samples were extracted, and quantitative reverse transcription (qRT)–PCR was performed, as previously described [15]. Phenol–chloroform was used to isolate total RNA from liver tissue (Isogen, Nippon Gene Co., Ltd., Tokyo, Japan) and then converted to cDNA with Moloney murine leukemia virus reverse transcriptase (Takara, Otsu, Japan). Quantitative reverse transcription was performed using an AriaMx Real-Time PCR system (g8830a, Agilent, Santa Clara, CA, USA). The sequences of primers used in this study were provided in a previous study [15].

#### *2.8. Selection of a Candidate Reference Gene*

In order to select a reference gene that is stably expressed and has low variability in the present experiment system, the stability of the five candidate housekeeping genes (Table 1) was validated using NormFinder (MOMA, Aarhus, Denmark). The relative quantification for qRT-PCR was performed by standard curve method.

**Table 1.** Sequence of primers for housekeeping genes tested with quantitative reverse transcription PCR.


#### *2.9. Statistical Analysis*

Data are presented as the mean ± standard deviation (SD), and one-way ANOVA and Tukey multiple comparison tests were used to compare differences between groups using the software package, Graph Pad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA). *p* < 0.05 was considered significant.

#### **3. Results**

#### *3.1. LF Prevents Steatohepatitis and Fibrosis in Cx32*Δ*Tg Rats*

We initially investigated the safety and chemopreventive effect of LF on NASH in a Cx32ΔTg–HFD–DMN rat NASH model. The dosage of LF in previous clinical studies was to reflect the selection of dosages in the present study [23]. A significant difference in body weights between control and LF-treated groups was not found. A dose-dependent change in organ weights was not noted, although liver weights were significantly increased in the LF100 compared to control group (Table 2).

Histological observation by H&E staining also indicated that LF did not induce any changes in kidneys. In the liver, treatments of a HFD and DMN induced diffuse deposits of fat droplets with hepatocellular ballooning and neutrophil infiltration in the lobule (Figure 1a). Lactoferrin treatment significantly reduced fat deposition, lobular inflammation, and ballooning injury of hepatocytes in a dose-dependent manner (Figure 1a–d and Table S1), resulting in decreased NAS (Figure 1e and Table S1). Bridging fibrosis and activated hepatic stellate cells (HSC) were visualized by Azan staining and α-SMA immunohistochemical staining, respectively, in a Cx32ΔTg–HFD–DMN rat NASH model (Figure 2a). The histological fibrosis score, percentages of the Azan-positive area (collagen), and percentages of the α-SMA-positive area (activated HSCs) were significantly reduced by LF in the NASH model (Figure 2a–d and Table S1).

**Table 2.** Body and various organ weights in connexin 32 dominant negative transgenic rats fed a high-fat diet and dimethylnitrosamine with or without lactoferrin (100 or 500 mg/kg/day) at week 17.


LF100, lactoferrin 100 mg/kg/day; LF500, lactoferrin 500 mg/kg/day. Dunnett's test \*: *p* < 0.05, \*\*: *p* < 0.01 vs. Control.

**Figure 1.** Preventive effect of lactoferrin on nonalcoholic steatohepatitis in rats. Connexin 32 dominant negative transgenic (Cx32ΔTg) rats were fed a high-fat diet (HFD), given an intraperitoneal injection of dimethylnitrosamine (DMN), and treated with lactoferrin (LF) for 17 weeks. (**a**) Representative histological findings of hematoxylin and eosin (H&E) stains in liver sections taken from Control, LF 100 mg/kg/day (LF100) or LF 500 mg/kg/day (LF500) rat groups. (**b**–**e**) Histopathological analysis of non-alcoholic steatohepatitis (NASH) was evaluated by severity scores for (**b**) steatosis, (**c**) lobular inflammation, (**d**) hepatocellular ballooning, and (**e**) a non-alcoholic fatty liver disease activity score (NAS). Data is shown as the mean ± SD, *n* = 15–16 per group, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 compared to the Control group.

Biochemical analysis of serum indicated that the level of AST, T-chol, and LDL-C in the LF–treated groups was lower than that in the control group and the level of glucose of the LF100 was significantly higher than that of the control group. However, there was no dose-dependent change in serum hepatic enzymes, proteins, glucose, or lipids (Table 3). These results indicated that LF administration prevented the development of steatohepatitis and fibrosis without any adverse effects observed in a rat NASH model.

**Figure 2.** Attenuation effect of lactoferrin on fibrosis in rat nonalcoholic steatohepatitis. Connexin 32 dominant negative transgenic (Cx32ΔTg) rats were fed a high-fat diet (HFD), given an intraperitoneal injection of dimethylnitrosamine (DMN), and treated with lactoferrin (LF) for 17 weeks. (**a**) Azan staining (upper panels) and α-smooth muscle actin (α-SMA; lower panels) immunohistochemical stains of liver sections from Control, LF 100 mg/kg/day (LF100), or LF 500 mg/kg/day (LF500) rat groups. (**b**) Azan staining was used to evaluate the fibrosis score and (**c**) percentage of fibrosis area. (**d**) α-SMA–positive area. Data is shown as the mean ± SD, *n* = 15–16 per group, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 compared to the Control group.

**Table 3.** Hepatic enzyme serum levels in connexin 32 dominant negative transgenic rats fed a high-fat diet and dimethylnitrosamine with or without lactoferrin (100 or 500 mg/kg/day) at week 17.


Alb, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; GLU, glucose; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; LF100, lactoferrin 100 mg/kg/day; LF500, lactoferrin 500 mg/kg/day; T-chol, total cholesterol; TP, total protein. Dunnett's test \*: *p* < 0.05 vs. Control.

#### *3.2. LF Tends to Decrease the Induction of Preneoplastic Lesions in Cx32*Δ*Tg Rats*

To explore the effect of LF on carcinogenic potential during the development of NASH, the formation of preneoplastic hepatic foci, namely GST-P–positive foci, was quantitated by immunohostochemistry. A combination of HFD and DMN treatment increased both the number and area of GST-P–positive foci in Cx32ΔTg rats, although the carcinogenic potential was weaker than that induced by MCDD plus diethylnitrosamine (DEN; Figure 3a–c) [10,15]. In contrast, both the number and area of GST-P–positive lesions tended

to be decreased by LF intake (Figure 3a–c and Table S1). In accordance with these results, LF may have the potential to reduce hepatocarcinogenesis in NASH.

**Figure 3.** Effect of lactoferrin on hepatocarcinogenesis in rat nonalcoholic steatohepatitis. Connexin 32 dominant negative transgenic (Cx32ΔTg) rats were fed a high-fat diet (HFD), given an intraperitoneal injection of dimethylnitrosamine (DMN), and treated with lactoferrin (LF) for 17 weeks. (**a**) Liver sections showing representative foci positive for glutathione S-transferase placental form (GST-P) from Control, LF 100 mg/kg/day (LF100), or LF 500 mg/kg/day (LF500) rat groups. (**b**) The number and (**c**) area of GST-P–positive hepatic foci. Data is shown as the mean ± SD, *n* = 15–16 per group.

#### *3.3. LF Down-Regulates mRNA Expression of Inflammatory Cytokines in Cx32*Δ*Tg Rats*

Previous studies, including ours, strongly indicated that expression of inflammatory cytokines associated with inflammation *(Tnf-α*, *Il-6*, *Il-18*, *Ifn-γ*, and *Il-1β*) and fibrosis (*Tgf-β1*, *Timp1*, *Timp2*, *Col1a1*, and *Ctgf*) correlated with histological NASH activity in human and rodent models [15,26–28]. Thus, we further quantitated their mRNA expression level using qRT–PCR. NormFinder analysis revealed that the stability value of *Gapdh* was the smallest among examined candidate housekeeping genes (Figure 4a). Therefore, we concluded *Gapdh* as the most stable gene and used it as a reference. As shown in Figure 4b, the inflammatory cytokines, *Il-6*, *Tnf-α*, *Il-18*, and *Il-1β*, were significantly down-regulated by LF compared with the control, and a dose-dependency was observed with *Il-6*, *Il-18*, and *Il-1β*. While not significant, *Ifn-γ* mRNA expression also tended to be decreased by LF (Figure 4b and Table S2). The mRNA expression of *Tgf-β1*, *Col1a1*, *Timp1*, *Timp2*, and *Ctgf* as fibrosis-related cytokines was also measured; *Timp2*, *Col1a1*, and *Tgf-β1* were significantly down-regulated by LF (Figure 4c and Table S2). These results suggests that down-regulation

of inflammatory cytokines by LF was involved in the attenuation of steatohepatitis and hepatic fibrosis in a Cx32ΔTg–HFD–DMN rat NASH model.

**Figure 4.** Down-regulation of inflammatory cytokines by lactoferrin in rat nonalcoholic steatohepatitis. Connexin 32 dominant negative transgenic (Cx32ΔTg) rats were fed a high-fat diet (HFD), given an intraperitoneal injection of dimethylnitrosamine (DMN), and treated with lactoferrin (LF) for 17 weeks. (**a**) NormFinder stability values for candidate housekeeping genes (*Gapdh*, *B2m*, *Actb*, *Ppia*, and *Gusb*). (**b**,**c**) mRNA levels of (**b**) pro-inflammatory cytokines (*Tnf-α*, *Il-6*, *Il-18*, *Ifn-γ*, and *Il-1β*) and (**c**) profibrotic cytokines (*Tgf-β1*, *Timp1*, *Timp2*, *Col1a1*, and *Ctgf*) in Control, LF 100 mg/kg/day (LF100), or LF 500 mg/kg/day (LF500) rat groups were measured using quantitative reverse transcription (RT)–PCR. Data is shown as the mean ± SD, *n* = 15–16 per group, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 compared to the Control group.

#### *3.4. LF Administration Reduces NF-κB Signaling in Cx32*Δ*Tg Rats*

Previous studies showed that NF-κB and JNK/SAPK signaling were switched on in a rat NASH model mediated by MCDD or HFD and DMN combined [10,15]. Therefore, we investigated how such signal transduction was altered by the administration of LF. Western blotting showed that elevated pNF-κB protein expression in NASH was significantly decreased by the administration of LF in a dose-dependent manner. In contrast, phosphorylated Mkk4 and Jnk, which belong to JNK/SAPK signaling, were not affected by LF, even though their upstream protein, Cdc42, was significantly reduced in LF–treated groups (Figure 5). Such results indicate that the inactivation of NF-κB, but not JNK/SAPK signaling, is involved in the preventive effect of LF against NASH development in rats.

given an intraperitoneal injection of dimethylnitrosamine (DMN), and treated with lactoferrin (LF) for 17 weeks. (**a**) Protein levels of nuclear factor (NF)-κB-related (NF-κB, phosphorylated (p)NF-κB, IκB-α) and SAPK/JNK (Cdc42, Mkk4, pMkk4, Jnk, and pJnk) signaling proteins in Control, LF 100 mg/kg/day (LF100), or LF 500 mg/kg/day (LF500) rat groups were assessed by western blotting. Each lane represents a protein sample from an individual rat. Phospho, phosphorylated. (**b**) Data is shown as the mean ± SD. \* *p* < 0.05, \*\* *p* < 0.01 compared to the Control group.

#### **4. Discussion**

In this study, we examined the chemopreventive effect of LF on NASH in a Cx32ΔTg– HFD–DMN rat NASH model. NASH is an internationally prevalent chronic liver disease that shows fatty accumulations in the liver, ballooning, and inflamed hepatocyte. Nonalcoholic fatty liver disease often occurs in adults as a complication of lifestyle-related diseases, although it has also been found in children and is increasing all over the world regardless of a country's industrialization level [3,29]. Eventually, continuous inflammation leads to fibrosis and progresses to cirrhosis and HCC. It is known that nearly 20% of NASH cases progress to liver cirrhosis and HCC, with or without liver cirrhosis [30,31]. Therefore, NASH is one of the most crucial targets for deterring liver cirrhosis and HCC.

Currently, the main treatments for NASH include an improvement in life-style, represented by diet and exercise therapies. In addition, existing drugs for other diseases might also be effective for countering NASH. For instance, for drug repositioning, insulin sensitizers, such as pioglitazone, improved hepatocyte injury and fibrosis in a randomized, placebo-controlled trial [32]. Ratziu et al. reported that rosiglitazone decreased liver steatosis, but an improvement in hepatocyte injury and fibrosis was not found [33]. Vitamin E [34,35] and drugs for hypercholesterolemia [36] have also been used as drug therapies for NASH but have not been established as standard treatments due to their less potent medicinal effects and possible side effects after long-term use [33,37,38]. Consequently, daily intervention with functional supplements, along with lifestyle modification, is considered essential in preventing NASH progression. Considering the pathogenesis of NASH, we hypothesized that the anti-inflammatory effects of LF would prevent this disease. Previous studies proposed that LF expression in the liver was decreased in highfructose, high-fat, or MCD-induced mouse NAFLD models [39]. In contrast, LF ameliorated HFD-induced hepatic steatosis and elevated the triglyceride level in mouse models [40–42]. In addition, the levels of hepatic triglycerides and visceral fat were decreased by LF and were positively correlated in ICR mice [43], indicating that LF has the potential to reduce fat accumulation in the liver.

In the present study, for the first time, the effect of LF on histological features of NASH, including steatosis, hepatocyte injury, and inflammation, was quantitated using scoring systems originally designed for humans [24,44]. Lactoferrin significantly improved steatosis in the liver but did not affect visceral fat weight in the rat NASH model. These findings indicated that LF might protect hepatic steatosis by moderating fatty acid metabolism in the liver or in adipose tissue. The balance of lipid metabolism in a whole body may determine the effect on the lipid environment in each organ. Hepatocyte injury and inflammation in NASH were also decreased in LF–treated groups. It is well-known that the inflammationassociated cytokines, *Il-6*, *Tnf-α*, and *Il-1β*, were up-regulated and involved in the evocation of chronic inflammation in the colon and liver [15,17]. As already demonstrated in the colon, down-regulation of these cytokines by LF was also induced in NASH in the present study. Altogether, LF may prevent steatohepatitis via a decrease of inflammatory cytokines.

Persistent chronic inflammation in NASH leads to increased fibrosis; similar to other chronic hepatitis diseases, the irreversible alteration of liver structure due to progressive fibrosis eventually leads to cirrhosis [45]. Therefore, fibrosis is one of the most important prognostic factors for patients with NASH. In accordance with previous studies, LF has the potential to suppress liver fibrosis induced by thioacetamide [46,47]; however, the effect on fibrosis during NASH has not yet been established. This is due to the fact that it is not easy to induce fibrosis with NASH over a short time period in an animal model. As shown in Figure 3a, advanced fibrosis when bridging between lobules, or a lobule and portal vein, was induced by LF in a Cx32ΔTg–HFD–DMN rat NASH model. Lactoferrin significantly decreased not only steatohepatitis but also the histological score and area of fibrosis in the model. The numbers of hepatic stellate cells with an active phenotype were increased in NASH and decreased in the livers of LF–treated rats. These novel findings indicate that LF prevents the liver fibrosis of NASH via the inactivation of HSC.

To date, the anti-tumor effect of LF on HCC was described in previous studies using a DEN-induced HCC model in rats or mice [48,49], but effects of LF on NASH-related hepatocarcinogenesis have not been established. The glutathione S-transferase placental form is a well-known marker for preneoplastic lesions in rat liver. Therefore, carcinogenic potentials in the liver can be measured by GST-P immunohistochemistry in the early phase of hepatocarcinogenesis [25]. The number and area of GST-P-positive foci in the liver tended to decrease in LF-treated groups, although a significant difference was not found (Figure 4). We previously induced GST-P-positive foci in a NASH model using DEN [10] or DMN [15]. Both the number and area of GST-P-positive foci induced by DMN were decreased compared to those induced by DEN, which might influence the lack of significant difference by LF in this study. The chemopreventive effect of LF on NASH-related hepatocarcinogenesis should be investigated in a future study.

Nuclear factor-κB signaling plays central roles in inflammation and fibrosis during NASH progression. Especially in regard to fibrosis, activation of NF-κB stimulates parenchymal cells, including Kupffer cells [50] and enhanced TGF-β1 signaling that is essential as a profibrogenic mediator [51]. Transforming growth factor-β1 signaling modulates HSC as an active phenotype [27,52]. However, other reports indicated that TGF-β1 induced NF-κB activation [53]. In the present study, LF treatment decreased activated HSC and prevented fibrosis in a rat NASH model. Furthermore, NF-κB activation and TGF-β1 up-regulation in the model were attenuated by LF. However, JNK, which is also an important signaling pathway for fibrosis, was not altered by LF administration. These results suggest that LF protected the development of fibrosis by inhibiting NF-κB and TGF-β1 signaling.

#### **5. Conclusions**

This study demonstrated that LF prevents steatohepatitis and fibrosis without any adverse effects in a Cx32ΔTg–HFD–DMN rat NASH model. Therefore, LF may be a potential preventive or therapeutic application for this disease.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nu14010042/s1, Table S1: Histopathology of NASH, fibrosis, and hepatocarcinogenesis in connexin 32 dominant negative transgenic rats fed a high-fat diet and dimethylnitrosamine with or without lactoferrin (100 or 500 mg/kg/day) at week 17; Table S2: mRNA level of inflammatory cytokines using quantitative reverse transcription PCR.

**Author Contributions:** Conceptualization and methodology: A.N.-I. and S.T. (Satoru Takahashi); validation, formal analysis, and investigation: Y.A., A.N.-I., K.X., M.K., H.K., Y.N., S.I., Y.M., S.T. (Shuji Takiguchi) and S.T. (Satoru Takahashi); data curation, writing of original draft preparation: Y.A. and A.N.-I.; writing by reviewing and editing: A.N.-I. and S.T. (Satoru Takahashi); resources: H.T., M.T. and S.T. (Satoru Takahashi); project administration, funding acquisition, visualization, and supervision: A.N.-I. and S.T. (Satoru Takahashi). All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by JSPS KAKENHI Grant Number 26460492 and 19K07509 to A.N-I.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Animal Care and Use Committee (ethic code: no. 19-025, approved on 24 September 2019) at Nagoya City University Graduate School of Medical Sciences.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors are sincerely grateful to Koji Kato and Junko Takekawa for excellent technical assistance with preparing tissue sections and immunohistochemical staining.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

#### **Abbreviations**


#### **References**


## *Article Sargassum plagiophyllum* **Extract Enhances Colonic Functions and Modulates Gut Microbiota in Constipated Mice**

**Pissared Khuituan 1,2, Nawiya Huipao 1,2, Nilobon Jeanmard 2,3, Sitthiwach Thantongsakul 2,3, Warittha Promjun 2,3, Suwarat Chuthong 2,3, Chittipong Tipbunjong 1,2 and Saranya Peerakietkhajorn 2,3,\***


**Abstract:** Constipation is a symptom that is widely found in the world's population. Various dietary supplementations are used to relieve and prevent constipation. Seaweed is widely used for its health benefits. In this study, we aimed to investigate the effects of *Sargassum plagiophyllum* extract (SPE) on functions of the gastrointestinal tract and gut microbiota. The results show that SPE pretreatment increased the frequency of gut contraction, leading to reduce gut transit time. SPE pretreatment also significantly increased the secretion of Cl<sup>−</sup> and reduced Na<sup>+</sup> absorption, increasing fecal water content in constipated mice (*p* < 0.05). In addition, the Bifidobacteria population in cecal contents was significantly higher in constipated mice pretreated with 500 mg/kg SPE for 14 days than in untreated constipated mice (*p* < 0.05). Our findings suggest that SPE can prevent constipation in loperamide-induced mice. This study may be useful for the development of human food supplements from *S. plagiophyllum*, which prevent constipation.

**Keywords:** brown algae; *Sargassum plagiophyllum*; constipation; gastrointestinal transit; gut microbiota; transepithelial transport

#### **1. Introduction**

Constipation is a health symptom that has been reported to affect approximately 8.2– 32.9% of the world's population [1,2]. Constipation is often defined as infrequent and/or difficult bowel movements with a hard, dry stool [3,4], and it can be brought on by reduced physical activity, insufficient fluid intake, medication, and depression [5]. The condition is associated with gut microbiota imbalances involving decreased numbers of Bifidobacteria and Lactobacilli, increased numbers of pathogens, and suppressed intestinal motility [6]. Bifidobacteria, Lactobacilli, and Enterococci were effectively used in the treatment of constipation [7–9], and previous studies revealed that the levels of these bacteria were decreased in irritable bowel syndrome with constipation [10,11]. Several studies also showed that Enterobacteriaceae were increased in the condition of chronic constipation [10,12]. The recommended treatments for constipation include osmotic laxatives, generally lactulose, magnesium oxide, or polyethylene glycol [13], but the overuse of osmotic laxatives can result in dehydration and electrolyte imbalance. Clearly, these laxatives, which are available over the counter, can be harmful if patients incorrectly use them.

Alternative treatments emphasize dietary management to ensure a sufficient intake of dietary fiber and fluids [14–16]. Moreover, some nutritional plant products have been reported to aid the management of constipation. The extracts of *Aloe ferox* Mill, agarwood (*Aquilaria sinensis* and *Aquilaria crasna*), *Liriope platyphylla*, and prunes can increase intestinal

**Citation:** Khuituan, P.; Huipao, N.; Jeanmard, N.; Thantongsakul, S.; Promjun, W.; Chuthong, S.; Tipbunjong, C.; Peerakietkhajorn, S. *Sargassum plagiophyllum* Extract Enhances Colonic Functions and Modulates Gut Microbiota in Constipated Mice. *Nutrients* **2022**, *14*, 496. https://doi.org/10.3390/ nu14030496

Academic Editor: Md Soriful Islam

Received: 14 December 2021 Accepted: 21 January 2022 Published: 24 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

motility, as well as the frequency and weight of stools. In Japan, the consumption of the seaweed *Ulva prolifera* gives effective relief to constipation sufferers [17]. The nutritional and pharmaceutical benefits of algae have been known for many centuries. Algae contain compounds that exert anti-inflammatory, antimicrobial, and antioxidant effects [18]. They also contain high amounts of dietary fiber, which has been widely used for the treatment of gastrointestinal disorders, including constipation, diarrhea, and ulcerative colitis [17–21]. Usually, the fiber component of algae principally comprises structural polysaccharides. A recent study reported that algal polysaccharides increased the populations of Bifidobacteria and Lactobacilli both in vivo and in vitro [22–24]. The large group of brown algae includes the macroalgal genus *Sargassum*, which is widely distributed along the coasts of the Gulf of Thailand and the Andaman Sea [25]. In the *Sargassum* species, the dominant polysaccharides include alginate, laminarin, and fucoidan [26]. The polysaccharides in *Sargassum* have been widely studied in pharmacological research, such as research on anti-obesity, anticancer, anti-inflammatory, antibacterial, and antiviral activities [27,28]. A previous study revealed that the components of *Sargassum plagiophyllum* were 68.69% carbohydrates (including 22.24% fiber), 9.05% protein, 0.88% lipid and 21.38% ash [29]. Fucoidan is a long-chain-sulfated polysaccharide found in *S. plagiophyllum*, which potentially reduces inflammation, and has antioxidant, antitumor, and anti-cholesterol activities [30,31]. Moreover, several studies revealed that *S. plagiophyllum* extract contains phenolic compounds and fucoxanthin, which have therapeutic activity, such as antioxidant, anti-inflammatory, anticancer, anti-obesity, and antidiabetic activities [32,33]. A recent study revealed that *S. plagiophyllum* extract also has antioxidant activity [34].

The present study aimed to investigate the effects of *Sargassum plagiophyllum* extract (SPE) on the changes in colonic functions and gut microbiota in a constipation model of mice. The gut transit time, colonic motility patterns, colonic smooth muscle contractility, electrolyte transport across cell membranes in the colon, and colonic microbiota composition were investigated.

#### **2. Materials and Methods**

#### *2.1. Sargassum plagiophyllum Extract (SPE) Preparation*

Adult-stage *Sargassum plagiophyllum* was collected from Lanta Island, Krabi, Thailand. The preparation of SPE followed the method of a previously described extraction of an algal sample [35]. Briefly, 1 g of finely ground dried *S. plagiophyllum* was added to 100 mL of distilled water and autoclaved at 121 ◦C for 20 min. The autoclaved *S. plagiophyllum* was centrifuged at 2220× *g* for 10 min, and the supernatant was collected and freeze dried to obtain SPE powder.

#### *2.2. Animals and Experimental Design*

Adult male ICR/Mlac mice (4–5 weeks old, 25–30 g) were obtained from the National Laboratory Animal Center, Mahidol University, Thailand. The mice were reared in a humidity- and temperature-controlled room (50–55% humidity and 25 ± 2 ◦C) and under 12 h light: 12 h dark photoperiod at the Southern Laboratory Animal Facility, Prince of Songkla University, Thailand. All mice had free access to food and water. All experiments were approved and guided by the Animal Ethics Committee of the Prince of Songkla University, Thailand (Project license number: MOE 0521.11/1555, Ref.68/2018).

The mice were divided into six groups (n = 5–6 in each group): a normal control, a constipation control, a positive control, and three treatments of SPE. The normal and constipation control groups were supplemented with 0.2 mL of distilled water. The positive control group was supplemented with 0.2 mL of 500 mg lactulose /kg of body weight, and the treatment groups were supplemented with 0.2 mL of SPE at 100, 500, and 1000 mg/kg of body weight. Lactulose and SPE were administered daily by oral gavage for two weeks. To prepare SPE and lactulose solutions for daily administration, SPE powder was freshly dissolved in distilled water. In all mice, except mice in the normal control group, constipation was induced by injection of 5 mg/kg loperamide (Lop) on day 12, day 13, and day 14 [34,36]. The body weight of each mouse was recorded every day.

On day 14, fecal pellets were collected for 4 h and then weighed and dried to calculate fecal water content. Gastrointestinal transit was also measured. The mice were anesthetized with 70 mg/kg thiopental sodium, and the small intestine, caecum, and colon were collected and dissected to study upper gut transit, colonic motility patterns, colonic smooth muscle contractility, epithelial transport in distal colon, and the composition of microbiota in cecal contents.

#### *2.3. Measurement of Gastrointestinal Transit*

To evaluate total gut transit time, mice were given a 0.1 mL Evans blue marker meal containing 5% Evans blue in 1.5% methylcellulose, and the time of the first blue pellet expulsion was recorded. A 3 mm glass bead was inserted into the colon (approximately 2 cm) using a plastic tip lubricated with petroleum jelly, and then the time to bead expulsion was recorded to observe the distal colonic transit time. For small intestinal transit, mice were gavage fed a 0.3 mL charcoal meal containing 10% *w*/*v* charcoal in 5% *w*/*v* gum arabic at 30 min before euthanasia. The euthanized mice were dissected, and transit (%) was calculated from the following equation [37]:

> Small intestinal transit (%) <sup>=</sup> the distance of charcoal meal total length of the small intestine <sup>×</sup> <sup>100</sup>

#### *2.4. Colonic Motility Pattern*

After dissection, the whole colon with natural fecal pellets was collected and placed in ice-cold Krebs solution (pH 7.4 with an osmolality of 289–292 mmol/kg H2O) in an organ bath with a Gastrointestinal Motility Monitor (GIMM) (Catamount Research and Development, St. Albans, VT, USA) and then continuously perfused at 10 mL/min with fresh oxygenated Krebs solution. The colon was allowed to equilibrate for 30 min in Krebs solution at 37 ◦C. The movement of fecal pellets was recorded using a video camera above the chamber, and then the images from each individual run were analyzed, and we constructed the spatiotemporal maps of motility using GIMM software [20]. The contraction patterns comprised propagating contractions and non-propagating contractions. The total number of spontaneous contractions was defined as the sum of propagating and nonpropagating contractions.

#### *2.5. Colonic Smooth Muscle Contractility*

To observe colonic smooth muscle contractility, the colon was first cleared of luminal content, and 1 cm colonic segments of proximal and distal colon were used and suspended in the direction of longitudinal smooth muscle fibers in a 10 mL organ bath containing oxygenated Krebs solution at 37 ◦C. To stimulate contraction, carbachol (Tocris Bioscience, Bristol, UK) was added to the Krebs solution in the organ bath in a cumulative fashion. The concentrations of carbachol progressed from 0.1 to 1 to 10 μM, without washing between increments. The amplitude of contraction (g) and frequency of contraction (times/min) were recorded with the PowerLab® System (AD Instruments, New South Wales, Australia) and analyzed with LabChart7 program software [20,37].

#### *2.6. Transepithelial Transport of Electrolytes across Cell Membranes in Distal Colon*

To observe the transport of Na<sup>+</sup> and Cl<sup>−</sup> across the epithelial cell membrane, 1 cm of distal colon tissue was opened and oriented as a flat sheet on an Ussing slider, which was placed in an Ussing chamber (Physiologic Instruments, San Diego, CA, USA) containing Krebs solution at 37 ◦C [21]. Carbogen was also included in this system to maintain the buffer at the physiological pH of 7.4 during the experiment. After that, transepithelial voltage (Vt) was recorded for 30 min as an equilibration period by injection of external current pulses (3 μA). To investigate Na<sup>+</sup> absorption by distal colon, 10 μM amiloride was added to the chamber at the apical membrane to inhibit Na+ absorption by the epithelial sodium channel (ENaC), and the change in Vt was then recorded for 10 min. Cl− secretion of Ca2+-activated Cl<sup>−</sup> channels (CaCC) was then induced by adding 100 μM of carbachol to the chamber at the basolateral membrane, and the change in Vt was again recorded for 10 min. Cl− secretion of the cystic fibrosis transmembrane conductance regulator (CFTR) was then induced by adding 10 μM forskolin at the basolateral membrane, and the changes in Vt were recorded for 10 min. Following Ohm's law, the transepithelial potential difference (Vte), transepithelial resistance (Rte), and equivalent short-circuit current (Isc) were calculated to represent the transepithelial transport of electrolytes in the collected distal colon [38,39].

#### *2.7. Composition of Colonic Microbiota Analyses*

Bacterial DNA of all samples were extracted from collected cecal content [40]. To amplify and detect bacterial 16S rRNA genes, qPCR was performed using LineGene 9600 Plus System (BIOER, Hangzhou, China) and SensiFAST™ SYBR® No-ROX Kit (Bioline). The following primer sets were used: FW 5 -CGATGAGTGCTAGGTGTTGGA-3 and RV 5 - CAAGATGTCAAGACCTGGTAAG-3 for total bacteria, LM26 5 -GATTCTGGCTCAGGAT GAACGC-3 and Bif228 5 -CTGATAGGACGCGACCCCAT-3 for Bifidobacteria, FW 5 - CGATGAGTGCTAGGTGTTGGA-3 and RV 5 -CAAGATGTCAAGACCTGGTAAG-3 for Lactobacilli, F-ent 5 - ATGGCTGTCGTCAGCTCGT-3 and R-ent 5 -CCTACTTCTTTTGCAA CCCACTC-3 for Enterobacteriaceae, and ECF 5 -AGAAATTCCAAACGAACTTG-3 and ECR 5 -CAGTGCTCTACCTCCATCATT-3 for Enterococci [41–45]. The following thermal cycling condition was used for all amplifications: 3 min at 95 ◦C, followed by 40 cycles of a two-step PCR reaction (5 s at 95 ◦C and 30 s at 60 ◦C) [40].

#### *2.8. Statistical Analysis*

All data are presented as means ± standard error (SE). The differences between groups were tested using one-way or two-way analysis of variance (ANOVA), followed by Bonferroni's test at α = 0.05 using GraphPad Prism 5 (version 5.01).

#### **3. Results**

#### *3.1. Effect of SPE Pretreatment on Body Weight, Fecal Water Content, and Gut Transit in Constipated Mice*

On day 14, the body weight of the mice in all treatment groups was not significantly different (Figure 1A, *p* > 0.05). Fecal water content was significantly lower in the constipation control group than in the normal control group (Figure 1B, *p* < 0.05). Fecal water content was significantly higher in the lactulose and SPE treatment groups than in the constipation control group (*p* < 0.05).

The effects of SPE treatment on gut transit were determined using the total gut transit time, small intestinal transit time, and evacuation time (Figure 2). The total gut transit time in the constipation control group was 503.60 ± 19.78 min. The total gut transit time in the normal control group was significantly shorter at 240.20 ± 26.59 min (Figure 2A, *p* < 0.001). The total gut transit time was also shorter in all three SPE treatment groups, and it was the shortest in the 1000 mg/kg SPE group (*p* < 0.001). The small intestinal transit time was not significantly different among all groups (Figure 2B). The evacuation time was slightly longer in the constipation control group (26.01 ± 3.40 min) than in the normal control group (25.02 ± 2.13 min), but it was not significantly different (Figure 2C, *p* > 0.05). However, the evacuation times were significantly shorter in the positive control (Lactulose + Lop) group and the 1000 mg/kg SPE treatment group than in the constipation control group (*p* < 0.05). Our results suggest that SPE pretreatment could shorten total gut transit time and evacuation time.

**Figure 1.** Effects of *Sargassum plagiophyllum* extract (SPE) pretreatment on body weight and fecal water content of constipated mice. (**A**) Body weight and (**B**) fecal water content of normal control group (control); constipation control group (SPE0 + Lop); 100, 500, and 1000 mg/kg SPE treatment groups (SPE100 + Lop, SPE500 + Lop, and SPE1000 + Lop, respectively); and positive control group (Lactulose + Lop). Symbols above the bars indicate significant differences from normal control or constipation control (# means *p* < 0.05 when compared with normal control group, and \*, \*\*, and \*\*\* mean *p* < 0.05, 0.01, and 0.001, respectively, when compared with constipation control group).

**Figure 2.** Effects of *Sargassum plagiophyllum* extract (SPE) pretreatment on gut transit of constipated mice. (**A**) Total gut transit time, (**B**) small intestine transit, and (**C**) evacuation time of normal control group (control); constipation control group (SPE0 + Lop); 100, 500, and 1000 mg/kg SPE treatment groups (SPE100 + Lop, SPE500 + Lop, and SPE1000 + Lop, respectively); and positive control group (Lactulose + Lop). Symbols above the bars indicate significant differences from normal control or constipation control (### means *p* < 0.001 when compared with the normal control group, and \*, \*\*, and \*\*\* mean *p* < 0.05, 0.01, and 0.001, respectively, when compared with the constipation control group).

#### *3.2. Effect of SPE Pretreatment on Colonic Motility Pattern in Constipated Mice*

The colonic motility pattern was investigated by determining the total number of contractions, the number of propagation contractions (peristalsis), and the number of non-propagation contractions (segmentation). Spatiotemporal maps were produced from an analysis of the contraction data using GIMM software (Figure 3). The total number of contractions was insignificantly higher in the normal control group than in the constipation control group (Figure 3A, *p* > 0.05), but the total number of contractions was significantly higher in the 500 mg/kg SPE treatment group than in the constipation control group (*p* < 0.05). Moreover, the number of propagation contractions was also significantly higher in the 500 and 1000 mg/kg SPE treatment groups than in the constipation control group (Figure 3B, *p* < 0.01). Non-propagation contractions were not significantly different among the groups (Figure 3C, *p* > 0.05).

**Figure 3.** Effects of *Sargassum plagiophyllum* extract (SPE) pretreatment on the colonic motility pattern of constipated mice. (**A**) Number of total contractions, (**B**) number of propagation contractions, and (**C**) number of non-propagation contractions of normal control group (control); constipation control group (SPE0 + Lop); 100, 500, and 1000 mg/kg SPE treatment groups (SPE100 + Lop, SPE500 + Lop, and SPE1000 + Lop, respectively); and positive control group (Lactulose + Lop). Symbols above the bars indicate significant differences from constipation control (\* and \*\* mean *p* < 0.05 and 0.01, respectively, when compared with the constipation control group).

#### *3.3. Effect of SPE Pretreatment on Colonic Smooth Muscle Contractility in Constipated Mice*

The amplitude and frequency of the contractions of the longitudinal smooth muscle fibers of the proximal and distal colon were observed to investigate the colonic smooth muscle contractility (Figure 4). The results revealed that the contractions of both the proximal and distal colon tended to be more frequent in the positive control (Lactulose + Lop) and SPE treatment groups. After adding 10 μM of carbachol, proximal colonic contractions occurred significantly less frequently in the constipation control (SPE0 + Lop) group (7.00 ± 0.73 times/min) than in the 500 mg/kg SPE treatment group (11.00 ± 1.63 times/min) (Figure 4A, *p* < 0.05). Distal colonic contractions were also significantly less frequent in the constipation control group (SPE0 + Lop) (9.33 ± 1.54 times/min) than in the normal control group (13.67 ± 1.12 times/min) (Figure 4B, *p* < 0.05). Even at 0.1 μM, contractions in the distal colon were significantly less frequent (6.83 ± 0.95 times/min) in the constipation control group than in the normal control group (11.83 ± 1.05 times/min) (*p* < 0.05).

**Figure 4.** Effects of *Sargassum plagiophyllum* extract (SPE) pretreatment on colonic smooth muscle contractility of constipated mice. Frequency and amplitude of contractions of (**A**,**C**) proximal colon and (**B**,**D**) distal colon of normal control group (control); constipation control group (SPE0 + Lop); 100, 500, and 1000 mg/kg SPE treatment groups (SPE100 + Lop, SPE500 + Lop, and SPE1000 + Lop, respectively); and positive control group (Lactulose + Lop). Symbols indicate significant differences from normal control or constipation control (# means *p* < 0.05 when compared with the normal control group, and \* means *p* < 0.05 when compared with the constipation control group).

The amplitude of the proximal colonic contractions showed a similar trend in all groups in that the amplitude of the contractions was highest at 10 μM of carbachol (Figure 4C). The amplitude of the proximal colonic contractions was lower in the constipation control group than in the normal control and positive control groups, as well as the 100, 500, and 1000 mg/kg SPE treatment groups, but there was no significant difference among all groups at all concentrations of carbachol (*p* > 0.05). The amplitude of the distal colonic contractions was highest at 1 μM of carbachol, but there was, again, no significant difference among all groups at all concentrations of carbachol (Figure 4D, *p* > 0.05).

#### *3.4. Effect of SPE Pretreatment on Transport of Electrolytes across Cell Membranes in Distal Colon of Constipated Mice*

The basal transport values (Vte, Rte, and Isc) of the distal colon were not significantly different among the groups (Table 1). However, these values did show significant

differences when the distal colon was exposed to amiloride, carbachol, and forskolin. The amiloride-induced Isc of the distal colon in the constipation control group (62.95 ± 1.77 <sup>μ</sup>Am/cm2) was significantly higher than the amiloride-induced Isc of the distal colon in the normal control (*p* < 0.001), positive control (*p* < 0.01), and SPE treatment (*p* < 0.001) groups (Figure 5A). In contrast, the carbachol-induced Isc of the distal colon in the constipation control group (19.46 ± 3.13 <sup>μ</sup>Am/cm2) was significantly lower than the carbachol-induced Isc of the distal colon in the normal control, positive control, and SPE treatment groups (Figure 5B, *p* < 0.001). The forskolin-induced Isc of the distal colon was also lower in the constipated control group (29.65 ± 1.92 <sup>μ</sup>Am/cm2) than in the normal control (*p* < 0.001), positive control (*p* < 0.01), and SPE treatment (*p* < 0.001) groups (Figure 5C).

**Table 1.** Transepithelial potential difference (Vte), transepithelial resistance (Rte), and equivalent short-circuit current (Isc) of distal colonic epithelium membrane of normal control, constipation control, positive control, and SPE-pretreated mice.


**Figure 5.** Effects of *Sargassum plagiophyllum* extract (SPE) pretreatment on transport of electrolytes across cell membranes of constipated mice. The charts show short-circuit current (Isc) responses to (**A**) amiloride, (**B**) carbachol, and (**C**) forskolin of distal colon in normal control group (control); constipation control group (SPE0 + Lop); 100, 500, and 1000 mg/kg SPE treatment groups (SPE100 + Lop, SPE500 + Lop, and SPE1000 + Lop, respectively); and positive control group (Lactulose + Lop). Symbols above the bars indicate significant differences from normal control or constipation control (### mean *p* < 0.001, respectively, when compared with the normal control group, and \*\* and \*\*\* mean *p* < 0.01 and 0.001, respectively, when compared with the constipation control group).

#### *3.5. Effect of SPE Pretreatment on Composition of Gut Microbiota in Constipate Mice*

Cecal contents were collected and weighed to estimate the numbers of total bacteria, Bifidobacteria, Lactobacilli, Enterobacteriaceae, and Enterococci in the cecum of mice from every control group and all SPE treatment groups. The cecal content weight of the mice in the 1000 mg/kg SPE treatment group (0.1584 ± 0.0117 g) was significantly higher than the cecal content weight of the mice in the constipation control group (0.0960 ± 0.0061 g) (Figure 6A, *p* < 0.01), but the numbers of total bacteria were not significantly different among groups (Figure 6B, *p* > 0.05). The number of Bifidobacteria was not significantly different between the constipation control and the normal control groups

(*p* > 0.05), but it was significantly higher in the 500 mg/kg SPE treatment group (1.33 ± 0.66 × 109 cells/g cecal content) than in the constipation control group (6.78 ± 3.42 × <sup>10</sup><sup>7</sup> cells/g cecal content) (Figure 6C, *p* < 0.05). The numbers of Lactobacilli, Enterobacteriaceae, and Enterococcus were not significantly different among groups (Figure 6D–F, *p* > 0.05). Our results suggest that pretreatment with 500 mg/kg SPE could modulate the composition of bacteria, especially Bifidobacteria, in the cecum of constipated mice.

**Figure 6.** Effects of *Sargassum plagiophyllum* extract (SPE) pretreatment on gut microbiota in constipated mice. (**A**) Weight of cecal contents and number of (**B**) total bacteria, (**C**) Bifidobacteria, (**D**) Lactobacilli, (**E**) Enterococci, and (**F**) Enterobacteriaceae in cecal contents of normal control group (control); constipation control group (SPE0 + Lop); 100, 500, and 1000 mg/kg SPE treatment groups (SPE100 + Lop, SPE500 + Lop, and SPE1000 + Lop, respectively); and positive control group (Lactulose + Lop). Symbols above the bars indicate significant differences from constipation control (\* and \*\* mean *p* < 0.05 and 0.01, respectively, when compared with the constipation control group).

#### **4. Discussion**

Our results suggest that SPE pretreatment increased the frequency of contractions in the colonic smooth muscle and effectively increased both the propagation contractions and the total contractions of the colon in constipated mice. Moreover, the frequency of the contractions of the constipation control mice was lower than that of the others; therefore, the total gut transit time of the constipation control mice was longer than that of the normal control and SPE pretreatment groups. This indicates that SPE is capable of preventing constipation by enhancing colonic contraction and reducing the gut transit time and evacuation time. The results of this study are consistent with the results of a previous study of the marine algae *Ulva* (*Enteromorpha*), which indicated that dried *Ulva* enhanced colonic contraction and reduced gut transit time in constipated mice [46].

A recent study found polysaccharides, such as alginates, laminarins, and fucoidans, in *Sargassum* [47]. These polysaccharides have been used as substrates for the fermentation and production of short-chain fatty acids (SCFAs) by gut microbiota [48]. The present study showed that the Bifidobacteria population in the cecal contents of constipated mice was significantly increased in mice pretreated with 500 mg/kg SPE. Bifidobacteria are beneficial microorganisms that stimulate the growth of butyrate-producing bacteria, such as *Faecalibacterium*, *Eubacterium,* and *Roseburia* [49]. Acetate, propionate, and butyrate have been shown to interact with the free fatty acid receptors 2 and 3 (FFA2 and FFA3) of enterochromaffin cells (ECs) to induce serotonin (5-HT) release and trigger peristalsis [50]. Our results show that the frequency of the total colonic contractions was higher and the total gut transit time was shorter in the SPE treatment groups than in the constipation control group. Pretreatment with SPE was therefore able to prevent constipation by enhancing colonic contractions and reducing the gut transit time and evacuation time. Therefore, SPE pretreatment prevented constipation in the loperamide-induced mice by promoting the beneficial bacteria that might enhance the butyrate production, which leads to increased colonic contractility. In this study, we observed four selected bacteria that were reported to be involved with constipation. For further studies, we suggest that 16S rRNA gene sequence analysis should be performed to observe the changes in the microbiota of SPE-treated mice.

Furthermore, the results of this study also suggest that SPE pretreatment increases fecal water content in constipated mice. Our results are consistent with those of previous studies that revealed a reduction in fecal water content and the secretion of water in the distal colon of constipated rats [51]. The feces of mice supplemented with lactulose and SPE in this study contained more water than the feces of mice in the constipation control group. This result supports the findings of studies of the marine algae *Ulva* and *Chondrus*. These algae induced the secretion of water into the colon and increased fecal water content [46,52,53]. Moreover, lactulose was also found to increase fecal water content by absorption [54].

The study of electrolyte transport across the epithelial cell membrane of the distal colon showed that the basal transepithelial potential difference (Vte), transepithelial resistance (Rte), and equivalent short-circuit current (Isc) were not significantly different among all groups. This result indicates that pretreatment with SPE did not affect the colonic tissue or the ion channels [39]. However, the functioning of the ion channels in the distal colon of constipated mice treated with SPE changed. Cl<sup>−</sup> secretion increased, and Na<sup>+</sup> absorption was inhibited in SPE-pretreated mice. These changes increased the fecal water content in these groups compared with the constipation control group. The increased Cl− secretion was confirmed by the increased Isc induced by carbachol in the distal colon of SPEsupplemented mice. SPE induced an influx of Ca2+ into gut epithelial cells, which activated the CaCC [55]. Moreover, the forskolin-induced Isc was also higher in the SPE pretreatment groups, indicating that cAMP increased in the cell and then activated CFTR and increased Cl− secretion [56]. *Sargassum* has been shown to contain flavonoids [57], which increase cyclic adenosine monophosphate (cAMP) in gut epithelial cells and then induce the release of Ca2+ from the endoplasmic reticulum to the cytosol via protein kinase A (PKA) [54,58]. Recent studies revealed that increased cellular Ca2+ levels are not only important for the activation of CaCC but that they also activate CFTR via the PI3K/Akt pathway [56,59]. Therefore, SPE pretreatment might increase fecal water content by increasing cellular Ca2+ levels to induce Cl− secretion in the colonic lumen.

High cellular Ca2+ levels also inhibited Na+ absorption and reduced water absorption in the colon [60]. In the present study, the amiloride-induced Isc of the distal colon of mice treated with SPE decreased. This result indicates that Na+ absorption in the distal colon reduced, and fecal water content therefore increased. In a recent study, it was found that goblet cell numbers on the villi of ileum increased in constipated mice pretreated with SPE [34]. This finding implied that mucus secretion in the ileum might also have increased, which supports our finding that fecal water content increased in constipated mice supplemented with SPE.

In conclusion, SPE is a natural supplement that enhances colonic contractility and increases the numbers of Bifidobacteria. Pretreatment with SPE reduced the gut transit time and the evacuation time of constipated mice. SPE also increased the secretion of Cl− and reduced Na+ absorption in the distal colon, leading to increased fecal water content. Therefore, SPE was able to prevent constipation.

**Author Contributions:** Conceptualization, S.P., P.K., C.T. and N.H.; methodology and data analysis, S.P., N.H., C.T. and P.K.; investigation, S.P., N.J., S.T., W.P. and S.C.; writing—original draft preparation, S.P.; writing—review and editing, S.P., P.K., C.T. and N.H.; project administration, S.P. and P.K.; funding acquisition, S.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the National Science, Research and Innovation Fund (NSRF) and the Prince of Songkla University (Grant No. SCI6405072S).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Animal Ethics Committee of the Prince of Songkla University, Thailand (ethical approval code: MOE 0521.11/1555 Ref.68/2018, date of approval: 28 December 2018).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data supporting the research for this study are available within the manuscript.

**Acknowledgments:** We are grateful to Supattra Pongparadorn and Jaruwan Mayakun from the Excellence Center for Biodiversity of Peninsular Thailand (Prince of Songkla University, Thailand) for the identification and collection of *Sargassum plagiophyllum*.

**Conflicts of Interest:** The authors declare no conflict of interest.

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## *Review* **Treatment of Glaucoma with Natural Products and Their Mechanism of Action: An Update**

**Ru Hui Sim 1, Srinivasa Rao Sirasanagandla 2, Srijit Das 2,\* and Seong Lin Teoh 3,\***


**Abstract:** Glaucoma is one of the leading causes of irreversible blindness. It is generally caused by increased intraocular pressure, which results in damage of the optic nerve and retinal ganglion cells, ultimately leading to visual field dysfunction. However, even with the use of intraocular pressure-lowering eye drops, the disease still progresses in some patients. In addition to mechanical and vascular dysfunctions of the eye, oxidative stress, neuroinflammation and excitotoxicity have also been implicated in the pathogenesis of glaucoma. Hence, the use of natural products with antioxidant and anti-inflammatory properties may represent an alternative approach for glaucoma treatment. The present review highlights recent preclinical and clinical studies on various natural products shown to possess neuroprotective properties for retinal ganglion cells, which thereby may be effective in the treatment of glaucoma. Intraocular pressure can be reduced by baicalein, forskolin, marijuana, ginsenoside, resveratrol and hesperidin. Alternatively, *Ginkgo biloba*, *Lycium barbarum*, *Diospyros kaki*, *Tripterygium wilfordii*, saffron, curcumin, caffeine, anthocyanin, coenzyme Q10 and vitamins B3 and D have shown neuroprotective effects on retinal ganglion cells via various mechanisms, especially antioxidant, anti-inflammatory and anti-apoptosis mechanisms. Extensive studies are still required in the future to ensure natural products' efficacy and safety to serve as an alternative therapy for glaucoma.

**Keywords:** glaucoma; herbs; traditional medicine; retinal ganglion cells; intraocular pressure

#### **1. Introduction**

Glaucoma is one of the leading causes of irreversible blindness, causing 6.6% of all blindness in 2010 [1]. According to the World Health Organization's (WHO) World Report on Vision, of the estimated 2.2 billion people having a vision impairment around the world, glaucoma affects an estimated 6.9 million people [2]. It has been further estimated that by 2040, approximately 111.8 million people worldwide aged between 40 and 80 years old will be affected by glaucoma [3]. Glaucoma is generally caused by intraocular pressure (IOP, >21 mmHg) build-up, resulting from blockage of intraocular fluid and aqueous humor drainage [4]. The elevated IOP progressively damages the retinal ganglion cells (RGCs) and optic nerve, causing visual field constriction that affects the peripheral field initially and the central vision field gradually [5]. Glaucoma patients require lifelong treatment and followup, and the disease has a significant negative impact on patients' quality of life in terms of anxiety, psychological well-being, daily life, driving and confidence in healthcare [6]. The main risk factors for glaucoma prevalence include age, family history with glaucoma, African American race, thinner central corneal thickness, pseudoexfoliation, pigment dispersion and myopia [7]. Additionally, an association between diabetes, hypertension, triglyceride levels and glaucoma were also identified [7,8]. Furthermore, genetic factors are also known to be risk factors for glaucoma, in which single-nucleotide polymorphisms in

**Citation:** Sim, R.H.; Sirasanagandla, S.R.; Das, S.; Teoh, S.L. Treatment of Glaucoma with Natural Products and Their Mechanism of Action: An Update. *Nutrients* **2022**, *14*, 534. https://doi.org/10.3390/ nu14030534

Academic Editor: Md Soriful Islam

Received: 23 December 2021 Accepted: 18 January 2022 Published: 26 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

numerous genes (e.g., myocilin, apolipoprotein E, X-ray repair cross-complementing group 1, zona pellucida glycoprotein 4) have been shown to be associated with an increased risk of glaucoma [9,10].

Glaucoma can be classified into two major types, i.e., open-angle (OAG) and angleclosure glaucoma (ACG), according to the physical obstruction of the aqueous humor drainage system, and the appearance of the iridocorneal angle and trabecular meshwork (TM) [11]. Alternatively, it can also be categorized as primary (idiopathic, not associated with other diseases or conditions) or secondary (attributed to underlying diseases or conditions, such as trauma, long-term medication, ophthalmic surgery, uveitis, necrotic tumors, diabetes or syndromic conditions) [11,12].

In primary OAG (POAG), aqueous humor drainage is obstructed or inadequate as there is an internal blockage within the TM [13]. In contrast, primary ACG (PACG) is characterized by the presence of a physical obstacle to the aqueous drainage as the iris is adhered to the cornea, obstructing the flow of aqueous humor to the TM and the uveoscleral drainage [12,14]. Symptoms appear more drastically in PACG, which results in a rapid reduction in the vision field, leading to total blindness. Other symptoms include ocular pain, headache, nausea, vomiting, multicolored halos and blurred vision [12]. Additionally, PACG is an ophthalmic emergency that requires immediate treatment to prevent the progression of irreversible ocular damage [12].

#### **2. Pathogenesis of Glaucoma**

The exact pathogenesis of glaucoma is complex and has not yet been fully elucidated. The potential mechanism involved in the neurodegeneration of glaucoma has been postulated to involve an amalgamation of mechanical, vascular, genetic and immunological factors.

#### *2.1. Mechanical Hypothesis*

The mechanical hypothesis explains the relationship between the IOP and RGC pathophysiology. The perforated lamina cribrosa (LC) is the weakest part of the sclera, and it is where the RGC axons pierce through the minute perforations to form the optic nerve, while the central retinal artery and vein pass through the LC via a larger central aperture [15]. Elevated IOP resulted from the imbalance between the production and drainage of aqueous humor, which led to the irreversible backwards bowing of the LC, in the process known as 'cupping' [16]. Optic nerve cupping is characterized by the remodeling of the extracellular matrix (ECM) and fibrosis in the LC [17]. Glaucomatous LC cells showed increased ECM gene expression and elevated intracellular calcium, which is known to promote proliferation, activation and contractility in fibroblasts via the nuclear factor of activated T cells/calcium signaling pathway [17]. This deformation damages the optic nerve and capillaries passing through the LC, disturbing the anterograde axonal transportation of RGCs, which then ultimately triggers visual field defects in glaucoma [16]. Furthermore, elevated IOP also resulted in activated pro-fibrotic pathway-induced ECM accumulation in the TM, leading to less efficient aqueous humor outflow, thereby causing further damage to the LC [18].

Ivers et al. [19] demonstrated that in experimental glaucoma monkeys, the first structural abnormality induced by elevated IOP was an increased anterior LC surface depth, followed by a decreased minimum rim width, and, lastly, a reduced retinal nerve fiber layer (RNFL) thickness. Different levels of increased IOP showed a remarkable effect on the visual field, best-corrected visual acuity and LC parameters (cup depth, LC depth, LC curvature index and prelaminar tissue thickness) [20]. Additionally, greater posterior displacement of the LC was significantly associated with a faster rate of loss of the RNFL [21]. RGC axonal degeneration and anterograde axonal transport deficits at the optic nerve head (ONH, the location where RGC axons converge to form the optic nerve and traverse the LC) precede the structural and functional loss of RGCs [22]. Disturbance of the RGC

anterograde axonal transport leads to the accumulation of metabolic waste in the cells and deprives the metabolic needs of the RGCs, subsequently causing their apoptosis [23].

In normal-tension glaucoma (NTG), patients also present with glaucomatous optic disc excavation, despite a normal IOP [24]. This suggests other risk factors are involved in the optic neurodegeneration of glaucoma. The LC serves as a barrier between the IOP within the eye, and the intracranial pressure within the cerebrospinal fluid-filled subarachnoid space surrounding the optic nerve; the pressure gradient between the LC is known as the translaminar pressure gradient (TLPG) [25,26]. The TLPG is higher in glaucoma patients, including NTG patients, and is associated with mechanical damage to the optic nerve fibers, anterograde axonal transportation disruption and altered blood flow, leading to glaucomatous damage [26–28].

#### *2.2. Vascular Hypothesis*

The blood flow of the ONH was significantly reduced in the eyes of pre-perimetric glaucoma patients, where there are characteristic glaucomatous changes in the optic disc, but without the presence of visual field defects [29,30]. POAG and PACG patients possess a lower capillary density, but with greater tortuosity and more dilated capillaries, compared to healthy individuals [31]. Similarly, both NTG and POAG patients showed lower retrobulbar velocities, and higher retinal venous saturation and choroidal thickness asymmetries, when compared to control subjects [32]. Decreased ocular blood flow was also shown to be correlated with structural glaucomatous progression, as indicated by retinal and optic nerve changes [33]. A recent retrospective longitudinal study revealed that reduced blood flow in the ONH precedes glaucomatous neurodegeneration in POAG patients [34]. The vascular hypothesis is thus based on the reduced perfusion pressure, faulty vascular autoregulation or loss of neurovascular coupling, which leads to optic nerve degeneration in glaucoma [35]. Due to the reduced ocular blood flow, this hypothesis proposes that the RGC axons suffer from oxygen and nutrient insufficiency, ultimately causing their degeneration. In a glaucoma rat model, ocular hypertension (OHT) led to selective hypoxia in the LC, which was associated with injured RGC axons, and axonal transport disruption [36]. This study also demonstrated upregulation of hypoxia-inducible enzyme heme oxygenase-1 (HO-1) and the anaerobic glycolytic enzyme lactate dehydrogenase, and increased generation of superoxide radicals in the retina and ONH, as well as the active subunit of the superoxide-generating enzyme NADPH oxidase, suggesting the involvement of oxidative stress [36]. Similarly, hypoxic RGCs were observed in young and aged glaucoma model DBA/2J (D2) mouse retinas, with a significant increase in the hypoxia-inducible factor-1α (HIF-1α) protein and reactive oxygen species (ROS), followed by a significant decrease in the antioxidant capacity and mitochondrial mass in the aged retinas [37].

#### *2.3. Oxidative Stress and Neuroinflammation in Glaucoma*

In accordance with animal studies, numerous studies have provided evidence of increased oxidative stress in glaucoma patients. In addition, blood and aqueous humor levels of oxidative stress-related molecular biomarkers, i.e., protein carbonyls and advanced glycation end products, significantly increased in glaucomatous samples compared with healthy controls [38]. Similarly, PACG patients presented with decreased serum levels of total antioxidant status (TAS) and superoxide dismutase (SOD), as well as increased levels of malondialdehyde (MDA), compared to healthy controls [39]. A meta-analysis further indicated that POAG patients had lower TAS in the blood and higher levels of SOD, glutathione peroxidase (GPX) and catalase (CAT) in the aqueous humor [40]. Oxidative stress is known to induce or dysregulate inflammation in the event of optic neurodegeneration from glaucoma.

Studies have shown that inflammation contributes to the disease progression of glaucoma. In glaucomatous human optic nerves, the number of CD163+ cells (a commonly used marker for anti-inflammatory macrophages involved in tissue repair and remodeling) was

significantly increased [41]. Systemic inflammatory status markers, i.e., the neutrophil-tolymphocyte ratio, platelet-to-lymphocyte ratio and systemic immune inflammation index, were significantly increased in POAG patients compared with the control group [42]. POAG patients exhibited a significant increase in various cytokines, i.e., serum interleukin (IL)-4, -6 and -12p70 and tumor necrosis factor-alpha (TNF-α), compared with the controls [43]. Similarly, elevated plasma TNF-α levels in patients with POAG and pseudoexfoliation glaucoma were detected [44,45]. Additionally, aqueous humor samples collected from chronic PACG patients showed significantly increased levels of eotaxin, macrophage inflammatory protein-1-alpha and interferon gamma (IFN-γ)-induced protein-10, and lower levels of TNF-α, IL-5, -9 and -17 and granulocyte-macrophage colony-stimulating factor, compared to the control group [46].

Glial cells in the retina, i.e., astrocytes, Müller cells and microglial cells having an important role in mediating inflammatory responses, have been shown to become reactive, leading to the production of inflammatory cytokines, causing further neuronal damage in glaucoma patients and experimental glaucoma models [47,48]. In general, cytokine signaling is linked to the inflammatory transducer nuclear factor-kappa B (NF-κB). In D2 mice, low energy-induced 5 adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in the retina and optic nerve triggered NF-κB p65 signaling, leading to increased pro-inflammatory TNF-α, IL-6 and nitric oxide synthase (NOS)-2 expression [49]. Injection administration of TGF-β2 increased IOP and ECM deposition in the TM of wildtype mice. In contrast, mice harboring a mutation in NF-κB blocked the effect, suggesting NF-κB is necessary for TGF-β2-induced ECM production and OHT [50]. Additionally, transgenic inhibition of astroglial NF-κB restrained the neuroinflammatory (reduced proinflammatory cytokine expressions, i.e., IL-1A, -1B, -2, -6, -10, -12 and -13, TNF-α and IFN-γ) and neurodegenerative outcomes (attenuated loss of RGCs and axons) of the eyes of an experimental OHT mouse model [51].

The current evidence indeed supports the contribution of neuroinflammation in the pathogenesis of glaucoma, but it is still not clear as to when neuroinflammation takes part in the sequence of pathological events in glaucoma. Neuroinflammation has been suggested to be secondary to the initial pathology (i.e., optic nerve crush injury) [52]. Optic nerve crush injury induced glial activation in the retina, which was significantly muted if RGC death was blocked by deletion of the Bax gene [52]. On the other hand, the inhibition of monocyte infiltration and microglial activation by X-ray treatment prevented neuronal damage and dysfunction in the ONH [53]. Nevertheless, immunomodulation has been shown to be beneficial in the progression of glaucomatous changes.

#### *2.4. Excitotoxicity of Glutamate*

In addition to the inflammatory response, glial cells in the retina also play a vital role in the function of the retina by providing homeostatic and metabolic support to the photoreceptors and retinal neurons [54]. Müller cells and astrocytes possess uptake and exchange systems for various neurotransmitters, including glutamate, via the glutamate/aspartate transporter (GLAST) in rodents, also known as the Na+-dependent high-affinity glutamate transporter-1 (EAAT-1) in humans [54,55]. Glaucomatous eyes have been shown to have decreased levels of EAAT-1, and the glutamate receptor subunit *N*-methyl-d-aspartate (NMDA)-R1 [56]. Furthermore, mice deficient in GLAST demonstrate spontaneous RGC loss and optic nerve degeneration without elevated IOP, suggesting the decrease in GLAST expression leads to glutamate excitotoxicity in the retina, as a possible pathogenesis of glaucoma [57].

As reviewed by others, perhaps the most accepted hypothesis involved in glaucoma pathogenesis currently may include the mechanical damage to the ONH induced by increased IOP, followed by vascular dysregulation (reduced ocular blood flow) and neuroinflammation (glial activation), which then disrupt axonal transport due to axonal mitochondrial function loss in the RGCs, ultimately leading to RGC axonal degeneration and

RGC cell death (Figure 1) [58–60]. However, the combination of mechanisms described earlier may vary greatly among different glaucoma patients [60].

**Figure 1.** Schematic diagram showing how oxidative stress, neuroinflammation, reduced ocular blood flow and excitotoxicity lead to subsequent pathological changes observed in glaucoma. The therapeutic potential of natural products against glaucomatous changes at various steps is shown with the symbol ×. RGC, retinal ganglion cell; IOP, intraocular pressure; ONH, optic nerve head.

#### **3. Glaucoma Research Models**

Numerous research models have been used to gain a considerable understanding of the pathogenesis of glaucoma, and to assess therapeutic approaches for glaucoma treatments [61–64]. In this section, we provide a brief overview of some of these models used by the studies presented in this review (summarized in Table 1); this helps to provide a better understanding of the discussions in the following sections.

There are several genetic glaucomatous animal models that present with an elevated or normal IOP. For instance, the D2 mouse presents a late-onset, chronic pigmentary glaucoma due to the high IOP that progresses with age, resulting from tyrosinase-related protein 1 (*Tyrp1*) mutation and a premature stop codon in glycoprotein non-metastatic melanoma protein B (*Gpnmb*), which collectively lead to anterior segment anomalies, iris atrophy, peripheral anterior synechiae and pigment dispersion [64,65]. In contrast, D2-*Gpnmb*<sup>+</sup> mice are the wild types for the *Gpnmb* mutation that do not develop increased IOP and glaucoma [66]. Alternatively, the *Vav2*/*Vav3*-deficient and connective tissue growth factor (βB1-CTGF) mouse models are other murine models of spontaneous glaucoma that present with elevated IOP, which leads to subsequent RGC loss [67,68]. Transgenic mice with a low overexpression of E50K mutant optineurin (E50K-OPTN) have been reported to present with enhanced axonal degeneration and decreased RGC survival, under normal IOP [69].

Glaucoma can also be induced in wild-type animal models by elevating the IOP experimentally. A high IOP can be achieved by blocking aqueous humor drainage with the injection of various substances (e.g., microbeads, hydroxypropyl methylcellulose and hyaluronic acid) into the anterior chamber [70–72]. Alternatively, injection of hypertonic saline into the episcleral vein [73], and cauterization [74] or laser photocoagulation [75–77] of the episcleral or limbal veins lead to TM scarring, which increases the resistance to aqueous humor drainage, resulting in an elevation in IOP. The elevated IOP in these models leads to varying degrees of RGC loss, glial activation and visual defects [75–78].

To investigate the role of excitotoxicity in glaucoma, RGC loss can be induced with the injection of NMDA intravitreally [79]. The optic nerve crush (performed by applying a crush injury to the optic nerve with a pair of cross-action forceps) or the complete optic nerve transection model causes all RGC axons to be damaged simultaneously, which results in the gradual loss of RGCs [80,81]. This non-IOP-related axonal degeneration research model is commonly used to assess the RGC neuroprotection properties of various substances [82]. The partial optic nerve transection model causes damage to only a portion of the RGC axons; thus, this model can study both primary (the death of RGCs whose axons have been cut off) and delayed secondary neurodegeneration (the death of RGCs whose axons are intact) [83]. Retinal ischemia/reperfusion (I/R) injury is known to be associated with glaucoma, and other eye diseases, and has been widely used as an animal model for OAG. I/R injury reduces retinal blood flow, which creates a state of retinal hypersensitivity to oxygen and other nutrients, precipitating severe oxidative and inflammatory damage when the circulation is subsequently reinstated (reperfusion) [84,85].


**Table 1.** Overview of glaucoma research models.

D2, DBA/2J; I/R, ischemia/reperfusion; IOP, intraocular pressure; NMDA, *N*-methyl-d-aspartate; RGC, retinal ganglion cell.

Numerous in vitro studies have utilized the RGC-5 cell line in glaucoma research to evaluate the neuroprotective properties of various supplements, including the studies reviewed here. However, it has now become clear that RGC-5 cells that were originally identified as immortalized rat RGCs were contaminated early in their development by the immortalized photoreceptor 661W cell line (RGC precursor-like cells) in the laboratory they originated from [86,87]. Therefore, the RGC-5 cells used by many of the studies described in the following section may not reflect the true phenotype of a mature RGC. Perhaps the use of primary RGCs from animal models would be better to investigate glaucomatous RGC responses to therapies in vitro [88].

In general, the various research models described represent only some aspects of glaucoma, thus each having different advantages over other models. It is important to use a suitable model based on the objective of the study.

#### **4. Natural Products Used for Glaucoma Treatment and Their Mechanism of Action**

In view of the role played by oxidative stress and neuroinflammation in glaucoma, the use of antioxidants may represent an alternative approach for glaucoma treatment. Currently, the mainstay of glaucoma treatment is the reduction in IOP, using IOP-lowering eye drops [89]. Other glaucoma treatments include laser trabeculoplasty and cyclodestruction, or surgical trabeculectomy, trabeculotomy, deep sclerectomy and viscocanalostomy, based on the European Glaucoma Society guidelines [90]. However, even when the IOP normalizes, the disease still progresses and affects visual function in some patients.

There has been significant research interest in complementary and alternative medicine (CAM), and it has been widely used in the treatment of glaucoma. In a survey involving a total of 1516 glaucoma patients in Canada, 10% of patients used CAM therapy specifically for glaucoma, and half of them believed that the treatments were beneficial [91]. Other recent surveys reported the prevalence of CAM usage to be 22% in Saudi Arabia and 67% in Palestine among eye patients [92,93]. The present review highlights recent studies on various CAMs used for the treatment of glaucoma.

#### *4.1. Gingko biloba* L.

*Ginkgo biloba* L. (GB) belongs to the Ginkgoceae family, and its leaves and seeds have been used for medicinal purposes for centuries [94]. With more than 70 different flavonoids having been identified in GB, it has been suggested to have broad-spectrum free radical scavenging activities [95]. Indeed, treatment with GB extract was able to increase the survival of a rat RGC line, following exposure to oxidative stress induced by hydrogen peroxide (H2O2) [96]. Furthermore, POAG patients treated with 120 mg of GB extract daily for at least 6 months demonstrated a lower rate of single-stranded DNA breaks in circulating leukocytes, indicating reduced oxidative stress [97].

Numerous clinical trials have also demonstrated that GB extract supplementation slows the progression of visual field damage and improves visual function in NTG patients [98,99]. However, Shim et al. [99] demonstrated that supplementation with 40 mg of GB extract, three times per day, showed no effect on the mean defect or contrast sensitivity in NTG patients, compared to those receiving placebo. Based on the vascular hypothesis of glaucoma pathogenesis, NTG patients receiving 80 mg GB extract tablets, twice a day for four weeks, showed a significant increase in ocular blood flow, volume and velocity, in comparison to the placebo group [100]. Furthermore, GB supplementation increased the radial peripapillary capillary vascular density in healthy subjects who received a 120 mg GB extract capsule daily for 4 weeks [101]. Table 2 summarizes clinical trials of natural products used for glaucoma treatment.

In animal studies, intraperitoneal injections of GB extract administered after optic nerve injury in rats were associated with a higher survival rate of RGCs [96,102]. This could be due to the anti-apoptosis property of GB, as demonstrated by the inhibition of apoptosis of RGCs via the modulation of mitogen-activated protein kinase (MAPK) signaling pathways, in the adult rat optic nerve injury model, following the retrobulbar injection of diterpene ginkgolides meglumine injection (DGMI, made from GB extracts, including ginkgolides A, B and K) [103]. Mechanistically, DGMI could inhibit cell apoptosis by inhibiting p38, JNK and Erk1/2 activation [103]. Additionally, GB extract-derived procyanidin B2 and rutin were shown to be able to protect human retinal pigment epithelial cells subjected to tert-butyl hydroperoxide-induced oxidative stress by modulating nuclear factor erythroid 2-related factor (Nrf)-2 and Erk1/2 signaling [104]. Another study proposed that P53, Bax, Bcl-2 and caspase-3/-9 could be considered as the core targets for GB extract

against apoptosis in H2O2-treated RGCs [105]. A summary of preclinical studies of natural products used for glaucoma treatment is provided in Table 3.

#### *4.2. Scutellaria baicalensis Georgi—Baicalin, Baicalein and Wogonin*

*Scutellaria baicalensis* Georgi, commonly known as Baikal skullcap or Chinese skullcap, is a widely used Chinese medicinal herb [106]. *S. baicalensis* extract and its three major active flavonoids, namely, baicalin, baicalein and wogonin showed low cytotoxicity and possessed neuroprotective, antioxidant, anti-inflammatory and anti-cancer properties [106–108].

Intragastric administration of 200 mg/kg of baicalein for 28 days significantly reduced IOP in a rat model of chronic OHT [109]. The decreased thickness of the RGC complex and the reduced nucleus of the RGC layer mediated by OHT were significantly ameliorated by baicalein treatment and associated with reduced apoptosis of RGCs by upregulating the expression of the anti-apoptotic protein Bcl-2 [109]. Additionally, baicalein protects RGCs against retinal ischemia via the downregulation of HIF-1α, matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGF), and upregulation of HO-1 [110].

The intraperitoneal administration of wogonin, 10 min after the establishment of the optic nerve crush rat model, reduced the loss of RGCs and inhibited RGC apoptosis [111]. The study also demonstrated the anti-inflammatory property of wogonin in preventing TLR4-NF-κB-mediated neuroinflammation, as indicated by the reduced gliosis response, microglial activation and pro-inflammatory cytokine (TNF-α, monocyte chemoattractant protein-1 (MCP-1), iNOS, IL-6 and -1β and cyclooxygenase (COX-2)) expressions in the retina following optic nerve crush [111].

Intraperitoneal administration of baicalin increased the number of RGCs and attenuated pathological changes (indistinct layer of retinas, decrease in the thickness of the RGC layer (GCL, a retinal layer where RGCs and displaced amacrine cells reside) and RGC density) in a model of episcleral venous occlusion with cauterization to establish a mouse model of glaucoma with chronic elevated IOP [112]. Baicalin treatment also inhibited autophagy and activated PI3K/AKT signaling in glaucoma mice, as PI3K/AKT signaling was shown to restrain the apoptosis and inflammatory response of RGCs in glaucoma development [112]. Additionally, treatment with baicalin significantly increased cell survival, reduced ROS production and inhibited pro-inflammatory factor IL-1α and endothelial leucocyte adhesion molecule-1 (ELAM-1) production in cultured human TM cells exposed to H2O2 [113].

#### *4.3. Coleus forskohlii (willd.) Briq.—Forskolin*

*Coleus forskohlii* (willd.) Briq. is a medicinal plant indigenous to India and Southeast Asia [114]. The leaves, roots and tubers of *C. forskohlii* are a rich source of a diterpenoid called forskolin, which acts as a second messenger cyclic adenosine 3 ,5 -monophosphate (cAMP) booster, via the direct stimulation of adenylate cyclase [114]. Studies have revealed that cAMP is important in regulating aqueous humor dynamics in the ciliary body and TM [115]. Indeed, a previous study has shown that forskolin perfused arterially at 30, 100 and 1000 nM caused a significant reduction in the rate of aqueous humor formation in an isolated bovine eye preparation [116]. This may explain the hypotensive effect of forskolin administration, as shown in a double-blind, randomized controlled trial where POAG patients treated with forskolin 1% *w*/*v* aqueous solution eye drops, at two drops thrice a day, for 4 weeks, showed a significant decrease in IOP [117,118].

In animal studies, a dietary combination of forskolin, homotaurine, spearmint and vitamins B1, B6 and B12 was able to protect against RGC loss in a rodent model of optic nerve injury [119] and hypertensive glaucoma [120]. Both studies demonstrated that the forskolin supplement mixture may counteract the inflammatory processes via the reduction in cytokine (iNOS, IL-6 and TNF-α) secretion, thereby leading to decreased apoptotic markers (Bax/Bcl-2 ratio and active caspase-3), finally sparing RGC death and the preservation of visual function [119,120]. However, in contrast to the clinical studies, the forskolin supplement mixture did not affect IOP elevation in glaucomatous rodents [120].

#### *4.4. Erigeron breviscapus (vant.) Hand. Mazz.—Scutellarin*

*Erigeron breviscapus* (vant.) Hand. Mazz. (DengZhanHua in Chinese) is a dicotyledonous plant in the Compositae chrysanthemum family found primarily in southwest China, especially in Yunnan [121]. It has been used in traditional Chinese medicine, for the prevention and treatment of cardiovascular diseases [121]. *E. breviscapus* supplements administered for 6 months showed no obvious adverse effects, with a significant decrease in the mean defect and an increase in the mean sensitivity, in POAG patients with a controlled IOP, demonstrating its partial protective effect on the visual field in glaucoma [122]. In chronic elevated IOP animal models, *E. breviscapus* oral supplements were shown to reduce IOP, improve impaired visual function, increase the RGC density and reduce RGC axonal degeneration caused by elevated IOP [123,124]. In RGCs, *E. breviscapus* extract was shown to suppress the outward potassium channel currents, which was suggested to be one of the key mechanisms behind *E. breviscapus*'s beneficial effects against glaucoma-induced RGC damage and visual impairment [125].

The flavonoid scutellarin is one of the major constituents of *E. breviscapus*. A 3-week oral scutellarin treatment ameliorated retinal thinning and visual deficits in an induced chronic OHT glaucoma model [126]. Scutellarin protected RGCs and reduced impaired retinal microglial cells by inhibiting NLRP3 inflammasome-mediated inflammatory reactions, which was associated with a reduced upregulation of apoptosis-associated speck-like protein (a caspase recruitment domain), cleaved caspase-1 and IL-18 and -1β following acute OHT [127].

#### *4.5. Lycium barbarum* L.

*Lycium barbarum* L., commonly known as goji berry or wolfberry, has been widely used in China to treat various diseases, i.e., blurry vision, abdominal pain, infertility, dry cough, fatigue, dizziness and headaches, and has been used as a potent anti-aging agent [128]. The most abundant component in goji berries is represented by carbohydrates, and isolated *L. barbarum* polysaccharides (LBPs) have been found to exert various pharmacological properties, i.e., neuroprotective, hypoglycemic, anti-cancer, immunomodulatory and antioxidant properties [129,130]. LBP supplementation has been shown to protect RGC survival and preserve retinal function in various glaucoma models, i.e., acute OHT [131,132], chronic OHT [133,134] and partial optic nerve transection [135]. In the partial optic nerve transection model, LBP pre-treatment for 7 days prior to the injury was shown to delay secondary degeneration of RGCs [136]. The study also reported LBP exerting its neuroprotective effects by inhibiting oxidative stress and the JNK/c-jun pathway, and by transiently increasing the expression of insulin-like growth factor-1, which is a known neurotrophic factor determining the survival of RGCs during the early stages of optic nerve injury [136].

LBP has been shown to protect RGCs against oxidative stress injury by inhibiting the generation of ROS and reducing the mitochondrial membrane potential following cobalt chloride (CoCl2)-induced hypoxia [137]. Additionally, LBP significantly promoted cell viability, reduced apoptosis and decreased cleaved caspase-3/-9 and ROS levels in human TM cells after H2O2 administration [138]. Alternatively, LBP treatment has been shown to promote M2 polarization of microglia and downregulate autophagy after partial optic nerve resection, which contributes to the delayed secondary degeneration of RGCs [139]. Other studies have also suggested that LBP provides neuroprotection to the RGCs and retina by inhibiting vascular damage, probably via the regulation of endothelin-1 (ET-1)-mediated biological effects [131,133]. In a recent study, LBP treatment also promoted blood–retinal barrier maintenance and survival of RGCs in acute OHT mice, which were mediated through the regulation of amyloid-β production and advanced glycosylation end product receptor expression [140]. Furthermore, *L. barbarum* ethanolic extracts reduced angiopoietin-like 7 protein (ANGPTL7) expression while increasing that of caveolin-1 in PC12 neuronal cells exposed to hydrostatic pressures, which was associated with decreased gene expressions of ECM proteins, i.e., MMP-2, MMP-9, collagen I and TGF-β [141]. Previous studies have indeed indicated that ANGPTL7 modulates the TM's ECM [142] and

MMP-mediated ECM turnover in the TM, which leads to a reduction in outflow resistance in the conventional outflow pathway, and to maintenance of IOP homeostasis [143].

LBP treatment significantly reduced neuronal death and glial activation in the retina following I/R injury [144,145]. Furthermore, LBP treatment was able to alleviate ischemiainduced retinal dysfunction (exhibiting greater b-wave and oscillatory potential responses) [144,146]. The antioxidant levels (glutathione, SOD and CAT) in the retina were significantly higher, while the MDA level was lower, in the submicron and blended *L. barbarum* extract-treated groups, compared to the control [146]. Further studies demonstrated that LBP exerted its neuroprotective effects via the activation of Nrf2 and an increase in HO-1 protein expression in the retina after I/R injury [145].

#### *4.6. Diospyros kaki* L.

Persimmon (*Diospyros kaki* L.), belonging to the family Ebenaceae, is a well-known fruit rich in carbohydrates, dietary fibers, vitamins, minerals, carotenoids, phenolic compounds and other bioactive phytochemicals [147]. In addition to its fruit, persimmon's leaves are also rich in flavonoids that exhibit antioxidant properties [148]. Pre-treatment of RGCs exposed to excessive oxidative stress and excitotoxicity with an ethanolic extract of persimmon leaves (EEDK) increased cell viability in a concentration-dependent manner [149]. Further studies revealed that the neuroprotective effect of EEDK was associated with decreased levels of apoptotic markers, i.e., poly (ADP-ribose) polymerase, p53 and cleaved caspase-3, and increased expression levels of antioxidant enzymes, i.e., SOD, GPX and glutathione S-transferase [149]. The same study demonstrated that EEDK treatment protects the retina and RGCs in a partial optic nerve crush mouse model [149]. Additionally, EEDK was also shown to reduce elevated IOP in a glaucoma mouse model, by regulating the soluble guanylate cyclase α-1 (sGCα-1, a primary regulator of vascular hypertension) signal [150].

#### *4.7. Tripterygium wilfordii Hook F.—Triptolide and Celastrol*

*Tripterygium wilfordii* Hook F., commonly known as thunder god vine, is a traditional Chinese medicine widely used to treat autoimmune and inflammatory diseases including rheumatoid arthritis, systemic lupus erythematosus and dermatomyositis [151]. Triptolide and celastrol are the predominant active phytochemicals isolated from this plant, which exhibit similar pharmacological activities, i.e., anti-cancer, anti-inflammatory, immunosuppressive, anti-obesity and anti-diabetic activities [152]. Triptolide treatment improved RGC survival via the inhibition of microglial activation in glaucoma models [153–155]. Additionally, triptolide treatment inhibited the expression of TNF-α and the nuclear translocation of NF-κB in an optic nerve crush model, suggesting that the neuroprotective effect of triptolide was attributed, partly, to its anti-inflammatory property [155]. Similarly, celastrol treatment also improved RGC survival in glaucoma models [156,157].

#### *4.8. Crocus sativus* L.*—Crocetin and Crocin*

Saffron (the dried stigma of *Crocus sativus* L.) is a spice that is widely used in food preparation, as a flavoring and coloring agent [158]. Referred to as the 'golden spice', saffron is the highest-priced aromatic medicinal plant in the world, with numerous pharmacological properties such as anti-cancer, anti-diabetic, anti-inflammatory, antioxidant, immunomodulatory, antifungal and antimicrobial properties [158]. Oral administration of saffron extract was shown to decrease microglial numbers and their activation following increased IOP, and this led to the prevention of RGC death [159]. A randomized interventional pilot study revealed that 30 mg/day saffron supplementation significantly reduced IOP in POAG patients, after 3 weeks of treatment [160].

More than 150 chemical compounds have been extracted from saffron, with crocin and crocetin being the two major active ingredients [161]. Intraperitoneal treatment with crocin can inhibit I/R-induced RGC death, and the effect of crocin may be mediated, partly, by its antioxidant action through the ERK pathway [162], or activation of the PI3K/AKT

signaling pathway [163]. Additionally, crocin protects RGCs against H2O2-induced damage by reducing ROS production and activating NF-κB [164]. Similarly, crocetin, an aglycone of crocin, prevented cell loss and apoptosis in the GCL in mice following NMDA- [165] and I/R-induced retinal damage [166].

#### *4.9. Curcuma longa* L.*—Curcumin*

Curcumin is a yellow pigment and an active component of the rhizome of *Curcuma longa* L., or turmeric [167]. It is known to possess antioxidant, anti-inflammatory, anti-cancer, anti-arthritis, anti-asthmatic, antimicrobial, antiviral and antifungal properties [167,168]. Considering that curcumin is a powerful antioxidant natural compound, it may represent another potential treatment to alleviate oxidative stress in glaucoma. Using an elevated IOP rodent model, curcumin treatment decreased the intracellular level of ROS and alleviated RGC apoptosis induced by oxidative stress [169]. In the same study, it was also observed that curcumin inhibited pro-apoptotic factors, such as caspase-3 and Bax, and upregulated the anti-apoptotic factor Bcl-2 [169]. In an ex vivo optic nerve injury model, thinning of retinal layers, especially the GCL, and strong RGC apoptosis were observed after 24 h post-injury, which correlated with a time-dependent increase in caspase-3 and -9 and pro-apoptotic marker levels, and a powerful activation of the JNK, c-Jun and ERK signaling (MAPK) pathways [170]. Curcumin prevented alterations in the apoptotic cascade and MAPK pathways, preserving RGC survival and retinal thickness [170]. In another experimental study in a rat retinal I/R injury model, curcumin supplementation in the diet for 2 days before I/R was able to protect the retina from ischemic injury [171]. Additionally, curcumin pre-treatment inhibited I/R-induced degeneration of retinal capillaries, which may occur through its inhibitory effects on injury-induced activation of NF-κB and signal transducer and activator of transcription 3 (STAT3), and on overexpression of MCP-1, a chemokine involved in the inflammatory response via recruitment of monocytes to injury sites [172].

Studies using TM cells exposed to H2O2-induced oxidative stress as an in vitro model observed that pre-treatment with curcumin reduced the production of intracellular ROS in a dose-dependent manner [173,174]. Curcumin alleviated oxidative stress-induced proinflammatory factors such as IL-1a, -6 and -8 and ELAM-1 and inhibited the apoptosis of TM cells [173]. Curcumin has also been shown to protect TM cells against oxidative stress and apoptosis via the Nrf2-keap1 pathway [174].

#### *4.10. Camellia sinensis* (L.) *Kuntze—Epigallocatechin-3-Gallate*

*Camellia sinensis* (L.) Kuntze, commonly known as green tea, is consumed as a beverage and is popular in China and Japan [175]. Green tea extract treatment administered orally to retinal I/R injury rats showed a higher number of surviving RGCs, and less apoptotic RGCs were observed [176]. Green tea extract treatment also reduced the increased protein expression (i.e., of apoptotic markers (activated caspase-3 and -8) and inflammation-related proteins (Toll-like receptor 4 (TLR4), IL-1β and TNF-α)) and p38 phosphorylation caused by the ischemic injury [176]. Additionally, green tea extract treatment led to suppression of activated microglia, astrocytes and Müller cells following lipopolysaccharide (LPS)-induced retinal inflammation in rats [177]. The green tea anti-inflammatory effects were associated with a reduction in the phosphorylation of STAT3 and NF-κB in the retina [177].

The major polyphenolic compounds contained in green tea are catechins, which include epigallocatechin-3-gallate (EGCG), which is also a powerful antioxidant, antiangiogenic and anticarcinogenic agent [175,178]. EGCG treatment was shown to preserve the RGC density in acute [179] and chronic elevated IOP rats [180], an optic nerve crush rat model [181], a retinal I/R injury rabbit model [182] and NMDA-induced excitotoxicity in rats [183]. Zhang et al. [179] reported that EGCG treatment significantly decreased inflammation-associated cytokine levels (IL-4, -6, -1β and -13, TNF-α and IFN-γ), and the proliferation rate of T lymphocytes. Furthermore, EGCG treatment inhibited the increase in the phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B cells

inhibitor, alpha (IκBα) and p65, leading to the suppression of NF-κB signaling pathway activation [179].

#### *4.11. Panax ginseng—Ginsenoside*

*Panax ginseng*, in the family Araliaceae, is considered as one of the most frequently employed medicinal herbs and functional foods [184,185]. In a randomized, placebocontrolled, crossover study, daily consumption of 3 g of Korean red ginseng (KRG) for 4 weeks was shown to improve daytime contrast sensitivity and ocular pain in glaucoma patients [186]. Following 8 weeks of KRG supplementation, glaucoma patients showed significant improvement in their tear film stability and total Ocular Surface Disease Index score, suggesting KRG improved dry eye syndrome in glaucoma patients [187]. Additionally, OAG patients receiving 1.5 g of KRG, orally 3 times daily for 12 weeks, showed significant improvement in the retinal peripapillary blood flow in the temporal peripapillary region [188].

Ginseng contains numerous phytochemicals such as ginsenoside (triterpenoid saponin), phenols and acidic polysaccharides [189]. These phytochemicals have been shown to protect RGCs. Total *Panax notoginseng* saponin treatment increased RGC survival and inhibited the cell apoptosis pathway induced by an optic nerve crush rat model [190]. Similarly, ginsenoside Rg1 treatment was able to reduce RGC damage in an ultrasound-targeted microbubble optic nerve damage rabbit model [191]. Furthermore, ginsenoside Rb1 protects RGCs against apoptosis caused by CoCl2-induced hypoxia and H2O2-induced oxidative stress [192].

#### *4.12. Cannabis sativa—Cannabinoids*

*Cannabis sativa,* commonly known as marijuana, is one of the most used psychoactive substances in the world [193]. The *C. sativa* plant contains more than 60 lipid-based cannabinoids, which are the signaling molecules of the endocannabinoid system; these include Δ-9-tetrahydrocannabinol (Δ9-THC), Δ-8-tetrahydrocannabinol (Δ8-THC), cannabidiol and cannabinol [194]. A reduction in IOP was observed in glaucoma patients associated with tachycardia, within the first 30 min after marijuana inhalation, with the duration of action limited to 4 h [195]. Similarly, Δ9-THC inhalation reduced IOP significantly from baseline in healthy adult subjects, detected from 40 min post-treatment and lasting up to4h[196].

In animal studies, a topically applied 2% Δ9-THC ophthalmic solution was shown to reduce IOP in clinically normal dogs [197]. To prolong the IOP reduction duration, the use of Δ9-THC-valine-hemisuccinate nanoemulsions, which help to increase absorption, produced a greater drop in IOP, compared to latanoprost and timolol in normal rabbits [198]. Similarly, a submicron emulsion of Δ8-THC treatment to normal and OHT rabbits also demonstrated a reduced IOP [199]. The IOP-lowering and RGC neuroprotective effects of cannabinoids have been shown to be mediated by CB1 cannabinoid receptors [200,201].

#### *4.13. Anthocyanins*

Anthocyanins, considered as flavonoids, are blue, red or purple pigments commonly found in the flowers, fruits and tubers of many plants [202]. Hence, the primary sources of anthocyanins are found in berries, currants, grapes and some tropical fruits [202]. Studies have demonstrated that anthocyanins provide numerous health benefits such as antioxidative and neuroprotective properties, prevention of cardiovascular diseases, anti-angiogenesis, anti-cancer, anti-diabetic, anti-obesity and antimicrobial activities and improved visual health [202,203].

OAG patients receiving supplementation of 50 mg of black currant anthocyanins daily for 24 months also showed a reduced IOP and improved visual field damage progression [204]. Black currant anthocyanin supplementation also enhanced blood flow to the ONH and its surrounding retina in OAG patients, with no changes in systemic conditions such as blood pressure and pulse rates observed [204,205]. Black currant anthocyanin supplementation also normalized the abnormal serum concentration levels of ET-1 in OAG

patients, suggesting that anthocyanins possibly affect the ET-1 receptor functions such as pharmacological reactivity and hypersensitivity [206].

The natural anthocyanins delphinidin, luteolinidin and peonidin were shown to be non-toxic to human retinal pigment epithelial (ARPE19) and RGC-5 cells, with luteolinidin and peonidin increasing the survival rates of the RGC-5 cells following exposure to H2O2 [207]. Administration of oral bilberry extracts rich in anthocyanins was shown to suppress RGC death following an optic nerve injury mouse model [208]. Bilberry extract administration increased chaperone molecule (Grp78 and Grp94) protein levels, an effect which may underlie the neuroprotective effect of bilberry extract after optic nerve crush [208]. In a model of light-induced retinal damage in pigmented rabbits, administration of bilberry anthocyanin extract at dosages of 250 and 500 mg/kg/day for 7 days significantly inhibited retinal dysfunction, as evidenced by the increased retinal outer nuclear layer thicknesses and lengths of the outer segments of the photoreceptor cells, compared to untreated rabbits with retinal degeneration [209]. Additionally, anthocyanin treatment attenuated the changes caused by light to the apoptotic proteins Bax, Bcl-2 and caspase-3 and increased the antioxidant enzyme levels (SOD, GPX and CAT), but it decreased the MDA level in the retinal cells [209].

#### *4.14. Resveratrol*

Resveratrol (trans-3,4 ,5-trihydroxystilbene) is a polyphenol found in berries, grapes, pomegranates and red wine [210]. It has been reported to possess a wide range of pharmacological effects, including cardioprotection, neuroprotection and anti-diabetic activity, due to its potent antioxidant and anti-inflammatory properties [210]. Resveratrol has been reported to increase oxidative stress markers, and the nitric oxide level in human glaucomatous TM cells, possibly by increasing endothelial nitric oxide synthase (eNOS) expression and reducing inducible NOS expressions [211]. In experimental glaucoma models, resveratrol treatment was shown to reduce RGC death [212,213]. Cao et al. [213] further demonstrated that intravitreal administration of resveratrol rescued RGCs by the decreased ROS generation in RGCs of a microbead-induced high-IOP mouse model. These studies support the antioxidant properties of resveratrol, which could be beneficial in glaucoma treatment.

Resveratrol protects RGC-5 cells against H2O2-induced apoptosis, by reversing H2O2 induced increased expressions of cleaved caspase-3/-9, production of ROS and the expressions of *p*-p38, *p*-ERK and *p*-JNK, proposing that resveratrol suppresses MAPK cascades to exert its neuroprotective effects in RGCs [214]. Additionally, resveratrol also mitigates retinal I/R injury-induced RGC loss, glial activation and retinal function impairment by inhibiting the HIF-1a/VEGF and p38/p53 pathways while activating the PI3K/AKT pathway [215–217].

In both the chronic OHT rat model and RGC-5 cells incubated under elevated pressure, RGCs showed apoptosis and mitochondrial dysfunction [218]. Resveratrol treatment improved the expression of proteins involved in mitochondrial biogenesis and dynamics, i.e., AMPK, Nrf-1, mitochondrial transcription factor A (Tfam), mitofusin 2 (mfn-2) and optic atrophy 1 (OPA1), which led to a decrease in RGC apoptosis, mitochondrial membrane potential depolarization and ROS generation [218,219]. Another recent study identified a potential mechanism involving the protective role of resveratrol in preventing ONH astrocyte dysfunction and degeneration, which would enable the astrocytes to continue providing structural and nutrient support to the optic nerve [220].

#### *4.15. Hesperidin*

Hesperidin is a flavanone commonly found in citrus fruits such as oranges, tangerines, lemons and grapefruits, known for its anti-inflammatory, antioxidant and anticarcinogenic properties [221]. The antioxidant profile of a novel supplement containing hesperidin, and two other food-derived antioxidants, i.e., crocetin and *Tamarindus indica* (tamarind), was assessed in a prospective, single-arm design trial involving 30 NTG patients receiving

the supplements for 8 weeks [222]. In patients with relatively high oxidative stress, the supplement significantly reduced the urinary 8-hydroxy-2 -deoxyguanosine (8-OHdG; a marker of oxidative DNA damage) level, and the biological antioxidant potential was also significantly elevated [222].

In an animal study, a single dose of oral hesperidin pre-treatment (25, 50 and 100 mg/kg) significantly reduced the increased IOP level in dextrose- and prednisolone acetate-induced OHT rats [223]. Additionally, hesperidin treatment increased the glutathione level in the aqueous humor and reduced morphological alteration in the ciliary bodies caused by elevated IOP [223]. Furthermore, hesperidin treatment ameliorated NMDA-induced retinal injury by suppressing oxidative stress [224] and excessive calpain activation [225] while also alleviating hypobaric hypoxia-induced retinal impairment through the activation of the Nrf2/HO-1 pathway [226].

#### *4.16. Caffeine*

Caffeine (1, 3, 7-trimethylxanthine) is a natural alkaloid commonly consumed through coffee, tea, carbonated soft drinks, energy drinks, chocolate and other cocoa-containing foods [227]. Caffeine acts as a central nervous system stimulant through its A1 and A2a adenosine receptor antagonist properties [227]. The effect of caffeine consumption on IOP was found to be controversial in the literature. Tran et al. [228] demonstrated a reduced IOP following 45 and 60 min consumption of caffeine in POAG patients, when compared to the water-drinking group. However, another study reported that 1% caffeine eye drops administered daily for a week showed no effect on IOP in POAG patients [229]. In contrast, healthy individuals receiving a single dose of a 4 mg/kg caffeine capsule showed an increase in IOP, with low-caffeine consumers reporting a more abrupt IOP increase compared to the high-caffeine consumers [230]. Further studies suggested the increase in IOP was associated with a reduction in the anterior chamber angle, which led to resistance to aqueous humor outflow [231]. Recent cross-sectional studies showed caffeine consumption was weakly associated with a lower IOP but was not associated with a decreased risk of developing glaucoma [232,233]. An in vivo study demonstrated a reduced IOP and prevention of loss of RGCs in the caffeine-drinking animals following laser-induced OHT in experimental rats [234]. However, the same study also reported that caffeine treatment did not ameliorate OHT-induced impairment in the RGC retrograde transport, although caffeine treatment appeared to partially attenuate axonal degeneration of the optic nerve induced by OHT [234]. Interestingly, caffeine drinking led to increased microglia reactivity, inflammatory response (IL-1β and TNF mRNA levels) and cell death following 24 h post-I/R injury in a mouse model, which were then reduced at day 7 postinjury [235]. Additionally, caffeine was shown to preserve the integrity of the blood–retinal barrier in LPS-treated ARPE19 cells, which can be considered as a new strategy to treat retinal degenerative diseases [236].

#### *4.17. Coenzyme Q10*

Coenzyme Q10 (CoQ10), or ubiquinone-10, is a natural lipophilic vitamin-like molecule with antioxidant and anti-inflammatory properties and is involved in the production and control of cellular bioenergy, pyrimidine synthesis, physicochemical properties of cellular membranes and gene expression [237,238]. It is predominantly found in animal organs (kidney, liver and heart) and is also present in meat, fish, soy oil and peanuts [238].

Treatment with CoQ10, either topically applied or supplemented in the diet, was shown to promote RGC survival by inhibition of RGC apoptosis in glaucoma models [239–241]. CoQ10 treatment has also been shown to inhibit glaucomatous mitochondrial alteration by the preservation of the mtDNA content and Tfam/oxidative phosphorylation (OXPHOS) complex IV protein expressions [239,240]. Furthermore, CoQ10 treatment inhibited the activation of astrocytes and microglial cells in the retina [239,240]. In a clinical study, CoQ10 and vitamin E eye drop administration in POAG patients for 12 months showed a beneficial effect on the inner retinal function (PERG improvement), with a consequent enhancement of the visual cortical responses (VEP improvement) [242]. Additionally, CoQ10 and vitamin E topical treatment increased RGC numbers, inhibited apoptosis and activated astrocytes and microglial cells in a mechanical optic nerve injury rat model [243].

#### *4.18. Vitamins*

A cross-sectional study involving a total of 2912 participants in the United States 2005–2006 National Health and Nutrition Examination Survey reported that supplementary consumption and serum levels of vitamins A and E were not associated with glaucoma prevalence [244]. A meta-analysis did not find an association between serum vitamin B6, vitamin B12 and vitamin D levels and different types of glaucoma [245]. Another recent systematic review concluded that blood levels of vitamins (A, B complex, C, D and E) did not demonstrate an association with OAG as well [246]. However, the same study reported that dietary intake of vitamins A and C showed a beneficial association with OAG [246].

The nicotinamide adenine dinucleotide (NAD+, an important metabolite for mitochondrial metabolism and oxidative stress protection) level in the retina of D2-*Gpnmb*<sup>+</sup> mice decreased with age [247]. Oral administration of vitamin B3 (nicotinamide, precursor of NAD+) was protective as both prophylaxis and an intervention of glaucoma, as shown by the reduced incidence of optic nerve degeneration, prevention of RGC soma and axonal loss and retinal nerve fiber layer thinning and preserved visual function [247,248]. In a crossover, randomized clinical trial involving 57 glaucoma patients, oral vitamin B3 supplementation for 6 weeks at 1.5 g/day, then for 6 weeks at 3.0 g/day, improved RGC function, but without affecting the IOP and RNFL thickness [249].


**Table 2.** Clinical trials evaluating natural products for glaucoma treatment.


**Table 2.** *Cont.*


**Table 2.** *Cont.*

8-OhdG, 8-hydroxydeoxyguanosine; BAP, biological antioxidant potential; ET-1, endothelin-1; HVF, Humphrey visual field; IOP, intraocular pressure; NTG, normal-tension glaucoma; OAG, open-angle glaucoma; OSDI, Ocular Surface Disease Index; PhNR, photopic negative; POAG, primary open-angle glaucoma; PERG, pattern electroretinogram; RGC, retinal ganglion cell; RNFL, retinal nerve fiber layer.

Previous studies have reported that serum vitamin D levels are significantly lower in glaucoma patients as compared to healthy subjects [250,251]. Additionally, the presence of polymorphisms in vitamin D receptors, e.g., the BsmI 'B' allele and TaqI 't' allele, was shown to be a relevant risk factor in the development of POAG [251]. Vitamin D deficiency subjects were reported to have higher, although not significant, IOP values compared to healthy individuals [252]. Treatment with 1α,25-dihydroxyvitamin D3 and its analog 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 through eye drops reduced the IOP in normal monkeys [253]. D2 mice treated with 1 μg/kg of 1α,25-dihydroxyvitamin D3, intraperitoneally for 5 weeks, showed improved RGC function (increased PERG and FERG amplitudes) and reduced RGC death, compared to vehicle-treated controls [254]. Additionally, the same study also reported decreased microglial and astrocyte activation, reduced inflammatory cytokines (IL-1β and -6, IFN-γ and CCL-3) and increased expression of neuroprotective factors (BDNF, VEGF-A and PlGF) in the 1α,25-dihydroxyvitamin D3 treatment group [254].

Induced OHT rats fed with a vitamin E-supplemented diet showed no difference in RGC cell death, compared to normal diet-treated rats [255]. However, the same study demonstrated that dietary vitamin E deficiency aggravated RGC apoptosis following induced OHT, which was found to be related to the increased level of lipid peroxidation [255]. In contrast, both topical and systemic α-tocopherol administration preserved the RGC numbers and retinal morphology in an optic nerve crush rat model [256].


Preclinicalstudiesnaturalproductsusedforglaucomatreatmentandtheirmechanism















#### **5. Challenges for Natural Product Application in Glaucoma Treatment**

The WHO has defined guidelines for evaluating the safety and efficacy of natural products, which is important to further supporting the use of CAM in the healthcare system [261]. This guideline provides general principles for both preclinical and clinical studies on evaluating herbal medicines, i.e., quality and preparation of plant materials, and general pharmacological, pharmacodynamic and toxicological analyses. Although the use of crude extracts from whole plants or a particular part of any herbal plant proves to be useful in the treatment of glaucoma, as described in this review, the identification and isolation of an active phytochemical may also be important, especially in the drug development process. Crude extracts contain a wide range of phytochemicals that may work synergistically or individually to provide a polypharmacy effect in the treatment of glaucoma [262]. Similarly, several studies have reported the use of a mixture of molecules to be effective in reducing IOP in POAG patients. Researchers may have difficulty in identifying the exact mechanism or compound responsible for such findings. For instance, oral administration of two tablets per day of a food supplement containing 150 mg of *C. forskohlii* extract (containing 15 mg forskolin), 200 mg of rutin, 0.7 mg of vitamin B1 and 0.8 mg of vitamin B2 for 30 days contributed to reducing IOP in POAG patients [263]. The same supplementation has also been shown to reduce ocular discomfort in POAG patients due to chronic use of multi-dose eye drops containing preservatives [264], and to prevent IOP spikes after neodymium:YAG laser iridotomy in patients at risk of POAG [265]. Additionally, supplementation with tablets containing *C. forskohlii* extract, homotaurine, carnosine, folic acid, vitamins of the B group and magnesium in POAG patients compensated by IOP-lowering drugs during a period of 12 months showed a significant further decrease in IOP and an improvement in the pattern electroretinogram amplitude at 6, 9 and 12 months, and foveal sensitivity at 12 months [266]. In another study, daily intake of a similar supplement for 4 months showed a decrease in IOP, improved light sensitivity and contrast sensitivity and a better quality of life in POAG patients [267]. Additionally, supplementation with French maritime pine bark/bilberry fruit extracts rich in anthocyanins to POAG patients for 4 weeks showed a reduced IOP [268].

Numerous eye drops of various classes, such as prostaglandin analogs, beta blockers, carbonic anhydrase inhibitors, adrenergic agonists, miotics and hyperosmotic agents, are often preferred over surgeries for the treatment of glaucoma [269]. One of the major issues in glaucoma treatment is patients' noncompliance, due to improper techniques of administering eye drops [270]. Another major issue is poor drug bioavailability across the blood–retinal barrier, limited retention capacity of the cul-de-sac (usually 7–10 μL, maximum 50 μL), rapid drainage of the medication caused by gravity and washout by tearing or through the nasolacrimal duct [271]. The use of various nanoformulations such as nanoparticles, nanoemulsions and nano lipid vesicles to transport phytochemicals may be able to increase the bioavailability of the drugs to the eye. For instance, baicalein loaded in trimethyl chitosan nanoparticles showed a longer pre-ocular retention time and improved baicalein bioavailability, compared to baicalein solution [272]. Davis et al. [258] reported the use of a curcumin-loaded nanocarrier formulation using D-α-tocopherol polyethene glycol 1000 succinate nanoparticles, with each particle measuring <20 nm in diameter. In an OHT rat model, topical application of curcumin nanocarriers administered twice daily for three weeks was shown to significantly reduce RGC loss, but not in the free curcumin treatment group [258]. Additionally, the same study showed that curcumin nanocarriers protected retinal cells against CoCl2-induced hypoxia and glutamate-induced toxicity in vitro, by significantly increasing cell viability [258]. Similarly, a chitosan–gelatin-based hydrogel containing curcumin-loaded nanoparticles decreased the inflammation (reduced expression of TNF and IL-1α and -6, associated with downregulated mitochondrial ROS production) and apoptosis levels (reduced TUNEL-positive cells and cleaved caspase-3 protein level) of human TM cells exposed to H2O2-induced oxidative stress [259]. Apart from curcumin, co-encapsulated resveratrol and quercetin in chitosan nanoparticles, and sodium alginatepoly (vinyl alcohol) electrospun nanofibers of forskolin showed an efficient IOP reduction

in adult normotensive rabbits [257,260]. These studies demonstrated that phytochemical nanoformulations hold promising results, promoting their use as an alternative to existing glaucoma eye drops in clinical practice.

Lastly, it is important to use a suitable methodology to address the objectives of a study. Numerous studies used the Bcl-2/Bax ratio to imply that the therapeutic substance influences the activation of the intrinsic apoptotic pathway in RGCs, as shown by the numerous studies which have been reviewed here. However, the concept that both Bcl-2 and Bax expressions are in a stoichiometric 1:1 balance in cells reflects the old 'rheostat' model of the Bcl-2 family's protein function, a hypothetical model that was debunked over two decades ago when it was shown that a 1:1 interaction of these proteins was a laboratory artifact [273,274]. Additionally, the predominant anti-apoptotic protein expressed in the retinal cells, including the GCL, is the long form of Bcl-X (Bcl-XL), which was found to be 16 times more abundant than Bcl-2 [275]. Furthermore, it is even questionable whether Bcl-2 is expressed in adult RGCs and may, in fact, be limited to Müller cells in the retina [276]. Therefore, the reporting of the Bcl-2/Bax ratio may not be a suitable marker to imply apoptosis in RGCs, and instead, the changes in Bcl-XL expression may correlate better with RGC apoptosis.

#### **6. Conclusions**

One of the most common causes of vision loss is glaucoma. Recent data have gained insight into glaucoma pathogenesis, which involves a complex interaction of LC cupping, insufficient ocular blood supply, oxidative stress and neuroinflammation. The use of natural products with antioxidant, anti-inflammatory and anti-apoptotic properties may prove to be beneficial in the treatment of glaucoma. Furthermore, natural products are easily available and are cost effective. Natural products have been shown to protect against RGC loss in in vitro and in vivo preclinical studies, as well as in clinical trials. The present review highlighted various natural products such as GBE, *L. barbarum*, *D. kaki*, *T. wilfordii*, saffron, curcumin, anthocyanin, caffeine, coenzyme Q10 and vitamins B3, D and E that confer neuroprotective effects on RGCs. Additionally, IOP has been shown to be reduced by treatment with marijuana, baicalein, forskolin, ginsenoside, resveratrol and hesperidin. GB, ginseng, anthocyanins and *L. barbarum* were reported to increase ocular blood flow in glaucoma. Additionally, caffeine administration has been shown to reduce IOP through its adenosine receptor antagonist properties. Although these may serve as alternative targets for glaucoma treatment other than IOP-lowering drugs, more evidence is required to warrant the recommendation of these novel targets. Admittedly, a few of these natural products have had no or limited clinical testing, restricting their potential use in the treatment of glaucoma. Nevertheless, it is important to ensure that the bioavailability and safety of these natural products are checked in well-designed randomized clinical trials to further determine their therapeutic potential in glaucoma.

**Author Contributions:** Conceptualization, R.H.S. and S.D.; writing—original draft preparation, R.H.S. and S.R.S.; writing—review and editing, S.L.T. and S.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

